- Email: [email protected]
DIHYDROPTERIDINE REDUCTASE DEFICIENCY DIAGNOSIS BY ASSAYS ON PERIPHERAL BLOOD-CELLS
1260
secondary to extracellular-fluid-volume depletion is not supported by our data. The stable body-weight, urinary sodium excretion, and, in the presence of severe renal failure, creatinine clearance, argue strongly against any significant change in plasma volume. Thiazide diuretics have an important place in the therapy of hypertension. In patients with essential thiazide diuretic results in an average fall in mean blood-pressure of between 9 mm Hg’2 and 25 mm Hg.13 The present study shows that chlorothiazide may also be an effective antihypertensive agent in patients with severe renal failure and produces an average fall in mean blood-pressure of 9
hypertension,
mm
treatment
with
a
standing p< 0-02).
Hg (supine p<0’01,
In
addition,
chlorothiazide has other beneficial actions; it appears to potentiate the action of other antihypertensive agents and its use is not usually associated with postural hypotension. No untoward reactions were noted in this study, although hypokalaemia (serum potassium <3.5 5 mmol/1) developed in 2 patients. The rise in blood urea, a recognised effect of thiazides, did not appear to be of clinical
significance. Requests for reprints should be addressed
to
B.J.
REFERENCES 1. Reubi FC. The action and use of diuretics in renal disease. Prog Cardiovasc Dis 1961; 3: 563-79. 2. Rubin AA, Beauregard SC, Hausler LM, Zitowitz L, Winbury MM. A nondiuretic benzothiadiazine with antihypertensive properties. Pharmacolo-
gist 1961; 3: 65. 3. Kincaid-Smith P, Fang P, Laver MC. A new look at the treatment of severe hypertension. Clin Sci Mol Med 1973; 44: 75-87. 4. Wilson IM, Fries ED. Relationship between plasma and extracellular fluid volume depletion and the antihypertensive effect of chlorothiazide. Circulation 1959; 20: 1028-36. 5. Finnerty FA, Davidov M, Kakaviatos N. Relation of sodium balance to arterial pressure during drug-induced saluresis. Circulation 1968; 37: 175-83. 6. Hollander W, Chobanian A, Wilkins RW. Relationship between diuretic and antihypertensive effects of chlorothiazide and mercurial diuretics. Circulation 1959; 19: 827-38. 7. Lauwers P, Conway J. Effect of long-term treatment with chlorothiazide on body fluids, serum electrolytes, and exchangeable sodium in hypertensive patients. J Lab Clin Med 1960; 56: 401-08. 8. Gifford RW, Mattox VR, Orvis AL, Sones DA, Rosevear JW. Effect of thiazide diuretics on plasma-volume, body electrolytes, and excretion of aldosterone in hypertension. Circulation 1961; 24: 1197-205. 9. Bourgoignie JJ, Catanzaro FJ, Perry HM. Renin-angiotensin-aldosterone system during chronic thiazide therapy of benign hypertension. Circulation 1968; 37: 27-35. 10. Beavers WR. Rat aorta and muscle electrolytes following chlorothiazide administration. Fed Proc 1960; 19: 99. 11. Shah S, Khatri I, Freis E. Mechanism of antihypertensive effect of thiazide diuretics. Am Heart J 1978; 95: 611-18. 12. Chalmers J, Tiller D, Horvath J, Bune A. Effects of timolol and hydrochlorothiazide on blood-pressure and plasma renin activity. Lancet 1976; ii: 328-31. 13. Hansen J. Hydrochlorothiazide in the treatment of hypertension. Acta Med
Scand 1968; 183: 317-21.
"The size of the pool from which we rescue our patients has the greatest impact on our results. It is in the unglamorous, undramatic, and frequently contentious area of prevention that the surgeon who makes part of his living from trauma might well return something to society ... improving safety for the local high-school football team, preaching on the dusty roads that lead to school groups or on the gustatory roads to Rotary luncheons, participating in local television shows on the value of helmets to cyclists, collecting and studying relevant data, or educating uninformed congressmen."-ALEXANDER J. WALK. In praise of surgical hedgehogs; trauma and the compleat surgeon. Bull Am Coll Surgeons 1979; 64: no.
10,4.
DIHYDROPTERIDINE REDUCTASE DEFICIENCY DIAGNOSIS BY ASSAYS ON PERIPHERAL BLOOD-CELLS RICHARD G. H. COTTON FRANK A. FIRGAIRA DAVID M. DANKS Genetics Research Unit, Royal Children’s Hospital Research Foundation and Department of Pœdiatrics, University of Melbourne, Australia
Human
peripheral lymphocytes, granulocytes, and platelets contain the enzyme dihydropteridine reductase. A simple procedure and assay for diagnosing dihydropteridine reductase on peripheral blood is described. In twelve controls the level of enzyme activity in a lymphocyte-platelet preparation was 29.5±4.8 (SD) nmol NADH oxidised/min/mg protein. Two heterozygotes for dihydropteridine reductase deficiency had activities of 6·4 and 10·3 nmol NADH oxidised/min/mg protein, respectively. Summary
Introduction DURING the past 5 years there has been rapid progress in the understanding of why progressive cerebral degeneration occurs in some patients with phenylketonuria despite early dietary treatment.l Clinical recognition of this new variant of phenylketonuria led to the demonstration of tetrahydrobiopterin (BH4) deficiency and consequent deficiency of cerebral production of neurotransmitters derived from tyrosine and tryptophan. The administration of these neurotransmitters, together with the usual dietary restrictions, was beneficial. 2.3 Claims that serum phenylalanine could be controlled by BH4 administration4 have subsequently been confirmed,5.6 and these cases can be treated with BH4 and neurotransmitters without dietary restriction. 6.7 These patients can be diagnosed by a single test-dose of BH4 (2 mg/kg orally) given to all babies with a positive Guthrie test before the phenylalanine restricted diet is started. Serum phenylalanine measured before and at 2 and 6 h after the dose8 falls to normal levels in patients with BH4 deficiency, whereas in those with classical phenylketonuria due to phenylalanine hydroxylase deficiency there is no change in serum phenylalanine.6.8 BH4 deficiency can be due to dihydropteridine reductase (DHPR) deficiency or be caused by a defect in denovo synthesis of BH4’ Until now DHPR deficiency has been proven by assay in liver biopsy specimens/’lO in cultivated fibroblasts,9.11 or in continuous lymphoid cell cultures transformed by Epstein-Barr virus." Support for diagnosis of a defect in BH4 synthesis has come from measurements of pterin precursors in blood and urine and from response of serum phenylalanine to administration of various precursors to BH44.66 In a baby shown to have BH4-responsive phenylketonuria the delay involved in measuring DHPR in cultivated cells is unacceptable, and liver biopsy has been necessary because DHPR had not been found in normal
peripheral lymphocytes or leucocytes.9 We describe here the application of an optimised ;assay" (utilising saturating concentrations of substrates and a higher [37°C] assay temperature) which shows that peripheral blood lymphocytes,neutrophils, and platelets have DHPR activity at levels useful for diag-
1261
nostic purposes. Activity is also described in red bloodcells. Genetic deficiency can be shown by tests on peripheral blood samples.
Methods
Separation of Peripheral Blood-cells Heparinised blood was separated by the discontinuous ’Ficoll-Isopaque’ density gradient technique of B0yum into two fractions: (A) ficoll-isopaque/plasma interface cells (lymphocytes, monocytes, and platelets), and (B) sedimented cells (granulocytes and erythrocytes). The cells found at the interface region (A) and the intermediate layer down to 2 mm from the erythrocyte meniscus were collected. This lymphocyte-platelet preparation was diluted and mixed with one volume of 0-15 mol/1 phosphate buffered saline (PBS), pH 7.4, and centrifuged at 5000 g for 30 min at 4°C. The cells were resuspended in an approximate fourfold volume of 0 -15 mol/1 KCI, freeze-thawed three times, and homogenised. The homogenate was then centrifuged at 12 000 g for 30 min at 4°C to pellet membranes. The supernatant was removed and assayed for DHPR activity.
Preparation of Purified Peripheral Blood-cells 50 ml of blood was obtained from two normal donors, and four major cell-groups were separated from the ficoll-isopaque
gradient. Lymphocytes were separated from the ficoll-isopaque/ plasma interface (A) by centrifugation at 700 g for 5 min and washed three times with PBS. The platelet-rich supernatant from the first 700 g lymphocyte preparation was collected. This was centrifuged twice more at 700 g to sediment contaminating red and white cells and then at 5000 g for 30 min to pellet the platelets. Granulocytes were separated from the erythrocytes by mixing of the ficoll-isopaque sedimented cells (B) with 2 volumes of PBS and 1 volume of 6% dextran T500 (Pharmacia) in PBS. The erythrocytes were allowed to sediment for 1 h after which the granulocyte-rich supernatant was collected and centrifuged at 700 g for 5 min. Contaminating erythrocytes in the pellet were lysed in 2 ml of distilled water for 1 min, and the granulocytes were then washed in PBS. This procedure was carried out three times. The erythrocytes sedimented in the granulocyte isolation step were washed three times with PBS. Total and differential cell-counts were done on each of the isolated cell suspensions to check purity. Extracts of the cells were prepared as described above and assayed for DHPR acti-
vity. DHPR
Assay
The modified assay for determination of DHPR activity in human cell-lines described recently" was used in this study. All assays were done in at least triplicate and at two concentrations of enzyme extract. Values shown in the tables are the averages of such multiple estimations.
Polyacrylamide Gel Electrophoresis Two non-denaturing gel disc gel electrophoresis
(1)
were used: done at 40C with 7.5% (w/v) to the method of Ornstein and
electrophoresis systems was
polyacrylamide gels according Davis,13 and (2) gradient gel electrophoresis (2.5% to 27% [w/v] polyacrylamide) was done as previously described.’a Staining for DHPR activity was done for 5 min with disc gels and 30 min for gradient gels by the procedure described by Cotton and Jennings.15 Protein was stained with Coomassie blue G250 and de-stained in 7% (v/v) acetic acid.
Results DHPR Levels in Lymphocyte-platelet Preparations Fraction (A) ficoll-isopaque-separated cells from a group of normal adult volunteers (aged 20-50 years) of both sexes were used to determine normal DHPR activity. Cell-counts and differentials showed that these cells were mainly lymphocytes and platelets (in 1/80 ratio). DHPR activity in these cells is shown in table i. In a blind experiment two blood samples obtained from the parents of a baby who died with DHPR deficiency", 16 were included with normal donor samples. The parents’ samples were identified correctly and the levels (% of mean) of activity were similar to those established previously in their cultured skin fibroblasts and continuous lymphoid cells (table I). DHPR activity was also measured in a group of
children being treated at this hospital (table I); no difference in DHPR activity could be observed between sexes and no trend with age was apparent in this or the adult group of controls. Investigation of sample handling showed that activity is retained in blood samples for 24 h at room temperature ; however, there is some erythrocyte contamination and a lowered platelet yield in the lymphocyte-platelet population obtained from such samples. The lymphocyte-platelet pellet may be stored frozen at —20°C; DHPR determination on two such samples 7 days later showed normal levels of activity. DHPR in Purified
Blood-cells
The DHPR activity in isolated lymphocytes, platelets, and granulocytes from two normal donors is presented in table n. The level of activity in the lymphocyte preparation is similar to that seen in human continuous lym-
phoid cells. 11I Attempts to assay erythrocyte extracts gave inconclusive findings since a very high level of non-specific oxidation of NADH occurred without the pterin substrate. No activity could be detected in serum from six normal controls.
Inhibition ofEnzymeActivity in Lymphocyte-platelet Preparations Extracts of these cells were incubated with known inhibitors of DHPR for 5 min at 37°C. Both 0-1 mmol/1 p-chloromercuribenzoic acid and 0-01 mmol/1 HgCl2 produced complete inhibition of activity. Preincubation with 0-1mmol/1 NADH for 10 min protected against this inhibition. These findings are in agreement with those observed by Cheema et al. 17 with sheep liver DHPR. The DHPR activity in these extracts depended on the presence of all the substrate and co-factor components in the assay reaction mixture.
Electrophoresis of DHPR from Human Blood-cells Electrophoresis in non-denaturing gels was done on samples of the cell extracts produced in this study and compared with purified human-liver DHPR.15 The gels were stained in situ for DHPR activity. Disc gels (separation according to charge) are seen in the accompanying figure. All extracts showed a prominent purple band
1262 TABLE I-DHPR ACTIVITY
(nmol NADH oxidised/mm/mg) IN
LYMPHOCYTE-PLATELET PREPARATIONS OF NORMAL CONTROLS AND PARENTS OF A CHILD WITH DHPR DEFICIENCY
I
I
I
I
iI
* Parents of patient who died of DHPR deficiency.3.16 Nos. in parentheses show activity as a percentage of the mean for that all type.
t Previously published.’1 t Samples of peripheral blood (3-5 ml) were obtained from eleven children (aged 4 mo - 8 yr age range; five females and six males) who had unrelated diseases.
which migrated with the same mobility as the purified human-liver enzyme. The gradient gel system (separation according to molecular size), showed a similar band of activity co-migrating with human liver DHPR. It is interesting to note that an erythrocyte extract gave a small activity band in both systems. Our finding of DHPR activity in mouse cell-lines derived from erythroblastomas (unpublished observation) suggests that this enzyme may even be found in erythrocytes. Discussion We have previously demonstrated the presence of DHPR activity in cultured continuous lymphoid cells," and in this study show that it is also present in peripheral white’blood-cells. It is difficult to be certain why Kaufman et al.9 and Abelson et al. 18 failed to detect DHPR activity in peripheral blood lymphocytes and leucocytes. We assume that differences in our assay procedures have led to our apparently conflicting results. Abelson et al.18 reported the presence of DHPR with a specific activity of 3 nmol NADH oxidised/min/mg protein in human platelets; this figure is only 11% of that observed in our study, which means that the assay procedure employed in their studies was less sensitive TABLE II-DHPR ACTIVITY IN ISOLATED BLOOD CELLS
I
*
Values are mean =SD in nmol NADH oxidised/min/mg; nos. in parentheses indicate no. of assays done. Identical activity was seen on cell extracts stored frozen and thawed 1 week later. t Non-specific oxidation of NADH precluded use use of this assay on
erythrocyte extracts.
Disc gel
electrophoresis of extracts from peripheral blood-cell preparations. (a’) and (a) represent purified human liver DHPR, 24 fLg and 0’2 {jLg, respectively; (b) lymphocyte extract 194 p.g; (c) platelet extract 248 p.g; (d) granulocyte extract 230 jig; (e) erythrocyte extract 400 jig, (f) and (f’) lymphocyte-platelet preparation (table t) 210 }ig. Samples (a’) and (f’) were stained for protein. Samples (a-f) were stained for DHPR activity. The faster migrating band in the erythrocyte extract (e) is haemoglobin.
possible explanation for their failure to detect actiin vity peripheral blood cell leucocytes. We believe that the activity shown in these peripheral blood leucocytes is truly due to DHPR because: 1. The enzyme activity was destroyed by appropriate inhibitors and by exclusion of components of the reaction mixture. 2. Heterozygotes for DHPR deficiency showed the expected result, these studies being done blind by the use of coded samples. 3. The level of residual activity observed in cells from these two heterozygotes was very similar to that found previously in their fibroblasts and continuous lymphoid cells. 4. The presence of DHPR has also been shown by a dif ferent approach consisting of gel electrophoresis followed by specific staining for enzyme activity. The band of activity moved to the same position as the purified enzyme from human liver on two different gel systems, which separated proteins according to charge and to molecular size, respectively. In designing a simple procedure for determination of the DHPR activity in a peripheral blood sample, we chose to use the lymphocyte-platelet preparation obtained by ficoll-isopaque separation. This technique is routinely available in most hospital laboratories and gives a good yield of DHPR-containing cells for a given
- a
1263
sample of blood. The difference in DHPR activity between lymphocytes and platelets is not great enough for variation in the relative numbers of these cells to disturb the result, at least for diagnosis of affected homozygotes (zero activity expected). Our results show that heterozygotes can also be identified. A cell-count on the preparation is suggested to check the lymphocyte/platelet ratio and to assist in the interpretation. The assay offered here is quick (1 day for cell preparation and enzyme determination) and simple. Moreover, only a small sample of peripheral blood is required, which eliminates the need for liver biopsies or the long delay incurred in waiting for cultured cells to grow. Activity is retained in heparinised blood samples for 24 h at room temperature or in cell pellets for more than 1 week at -20°C. Transport of samples to a specialist laboratory is therefore feasible. We thank Dr G. Tauro and Dr C. Hosking and their staff for their This work was supported partly by the National Health and Medical Research Council of Australia. F. A. F. was the recipient of a Commonwealth postgraduate research award.
help.
Requests for reprints should be addressed to D.M.D., Genetics Research Unit, Royal Children’s Hospital Research Foundation, Parkville, Victoria 3052, Australia.
REFERENCES
Clayton BE, Wolff OH. New variant of phenylketonuria with progressive neurological illness unresponsive to phenylalanine restriction.
1. Smith I,
Lancet 1975; i: 1108-11. 2. Bartholomé K, Byrd DJ. L-dopa and 5-hydroxytryptophan therapy in phenylketonuria with normal phenylalanine hydroxylase. Lancet 1975; ii: 1042. 3. Danks DM, Bartholomè K, Clayton BE, et al. Malign ant hyperphenylalaninæmia—current status (June, 1977). J Inher Metab Dis 1978; 1: 49-53. 4. Danks DM, Cotton RGH, Schlesinger P. Variant forms of phenylketonuria. Lancet 1976; i: 1236. 5. Schaub J, Daumling S, Curtius H-Ch, et al. Tetrahydrobiopterin therapy of atypical phenylketonuria due to defective dihydrobiopterin. Arch Dis Child 1978; 53: 674-76. 6. Curtis H-Ch, Niederwieser A, Viscontini M, et al. Atypical phenylketonuria due to tetrahydrobiopterin deficiency. Diagnosis and treatment with tetrahydrobiopterin, dihydrobiopterin and sepiapterin. Clin Chim Acta 1979; 93: 251-62. 7. Niederwieser A, Curtius H-Ch, Bettoni O, et al. Atypical phenylketonuria caused by 7,8-dihydrobiopterin synthetase deficiency. Lancet 1979; i: 131-33. 8. Danks DM, Cotton RGH, Schlesinger P. Diagnosis of malignant hyperphenylalaninæmia. Arch Dis Child 1979; 54: 329-30. 9. Kaufman S, Holtzman NA, Milstein S, Butler IJ, Krumholz A. Phenylketonuria due to a deficiency of dihydropteridine reductase. N Engl J Med
1975, 293: 785-90. 10. Brewster TG, Moskowitz MA, Kaufman S, Breslow JL, Sheldon M, Israel FA. Dihydropteridine reductase deficiency associated with severe neurologic disease and mild hyperphenylalaninæmia. Pediatrics 1979; 63: 94-99. 11. Firgaira FA, Cotton RGH, Danks DM. Human dihydropteridine reductase. A method for the measurement of activity in cultured cells, and its application to malignant hyperphenylalaninemia. Clin Chim Acta 1979; 95: 47-59. 12. Bøyum A. Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest 1968; 21: (suppl 97). 13. Ornstein L, Davis BJ. Disc electrophoresis, I and II. Distilation Products Industries, Rochester, 1962. 14. Cotton RGH, Jennings I. Affinity chromatography of phenylalanine hydroxylase. The structure of a ptendine adsorbent. Eur J Biochem 1978; 85: 357-63. 15. Cotton RGH, Jennings I. A napthoquinone adsorbent for affinity chromatography of human dihydropteridine reductase. Eur J Biochem 1978; 83: 319-24. 16. Danks DM, Schlesinger P, Firgaira F, et al. Malignant hyperphenylalaninemia—clinical features, biochemical findings and experience with administration of biopterins. Pediatr Res 1979; 13: 1150-55. 17. Cheema S, Soldin SJ, Knapp A, Hofmann T, Scrimgeour KG. Properties or purified quinonoid dihydropterin reductase. Can J Biochem 1973; 51: 1229-39. 18. Abelson TH, Gorka C, Beardsley PG. Identification of dihydropteridine reductase in human platelets. Blood 1979; 53: 116-21.
DETECTION OF VIRUS-ASSOCIATED ANTIGEN IN SERUM AND LIVER OF PATIENTS WITH NON-A NON-B HEPATITIS* L. VITVITSKI A. M. PRINCE
C. TREPO B. BROTMAN
INSERM U 45 et Centre d’Epidémiologie CNRS LP 005440, Hôpital Edouard Herriot, Lyons, France; Virology Laboratory, Lindsley F. Kimball Institute, New York Blood Center, New York, U.S.A.; and Vilab II, Liberian Institute ofBiomedical Research, Robertsfield, Liberia
Summary
In a search for serological markers of non-A non-B (NANB) hepatitis, sera
from
repeatedly transfused and convalescent patients assayed by immunodiffusion against sera from 12 patients with early acute NANB hepatitis. A new antigen/antibody system distinct from HBsAg was demonstrated in 8 cases. To assess the specificity of the test, serial sera from 17 patients with acute hepatitis of known ætiology (10 due to hepatitis-B virus, 4 to hepatitis-A virus, 3 to drugs) were tested twice a month, together with sera from 14 NANB patients obtained during a prospective post-transfusion study. NANB antigen (Ag) was detected in at least one sample from 12 of the 14 NANB patients (86%) but in none of the other groups. NANB Ag appeared after or just before elevation of transaminase levels and was cleared before they fell to normal. 4 of 5 patients who showed seroconversion to NANB antibody (Ab) had transient hepatitis. In were
contrast, the alanine aminotransferase value returned to normal in only 1 of the 5 with persistent NANB antigenæmia during 6 months’ follow-up. NANB Ag was also demonstrated by immunodiffusion in liver extracts from patients with chronic NANB hepatitis with antigenæmia. Fluorescein-isothiocyanate-labelled gammaglobulins with strong NANB Ab activity revealed specific nuclear fluorescence in foci of hepatocytes on cryostat sections of these livers but in none of 6 control human livers. The results suggest that the antigen and antibody are specifically linked to NANB hepatitis of long incubation period.
Introduction THE development of sensitive serological assays for hepatitis A and B has led to the recognition of a new category of viral hepatitis known as non-A non-B (NANB). Successful transmission of the disease to chimpanzees confirmed its infectious nature.’-5 More than one virus may be involved. 6.7 NANB hepatitis may account for up to 90% of all cases of post-transfusion hepatitis8.9 as well as for a significant proportion of spor-
adic
cases.
The causative viruses have
not
been identi-
fied, although various particles have been implicated.’O-" The finding that NANB infections are characterised by long-lasting virxmia and a tendency to progress to chronic hepatitis encouraged a search for possible circulating antigens which could be used as markers of NANB viruses. Shirachi et al.,13 using immunodiffusion, and Prince et aI.,5 using radioimmunoassay, have reported the existence of such an antigen/antibody sys*This paper was presented in part at the International Symposium on Hepatitis, Munich, April 5-7, 1979.
secondary to extracellular-fluid-volume depletion is not supported by our data. The stable body-weight, urinary sodium excretion, and, in the presence of severe renal failure, creatinine clearance, argue strongly against any significant change in plasma volume. Thiazide diuretics have an important place in the therapy of hypertension. In patients with essential thiazide diuretic results in an average fall in mean blood-pressure of between 9 mm Hg’2 and 25 mm Hg.13 The present study shows that chlorothiazide may also be an effective antihypertensive agent in patients with severe renal failure and produces an average fall in mean blood-pressure of 9
hypertension,
mm
treatment
with
a
standing p< 0-02).
Hg (supine p<0’01,
In
addition,
chlorothiazide has other beneficial actions; it appears to potentiate the action of other antihypertensive agents and its use is not usually associated with postural hypotension. No untoward reactions were noted in this study, although hypokalaemia (serum potassium <3.5 5 mmol/1) developed in 2 patients. The rise in blood urea, a recognised effect of thiazides, did not appear to be of clinical
significance. Requests for reprints should be addressed
to
B.J.
REFERENCES 1. Reubi FC. The action and use of diuretics in renal disease. Prog Cardiovasc Dis 1961; 3: 563-79. 2. Rubin AA, Beauregard SC, Hausler LM, Zitowitz L, Winbury MM. A nondiuretic benzothiadiazine with antihypertensive properties. Pharmacolo-
gist 1961; 3: 65. 3. Kincaid-Smith P, Fang P, Laver MC. A new look at the treatment of severe hypertension. Clin Sci Mol Med 1973; 44: 75-87. 4. Wilson IM, Fries ED. Relationship between plasma and extracellular fluid volume depletion and the antihypertensive effect of chlorothiazide. Circulation 1959; 20: 1028-36. 5. Finnerty FA, Davidov M, Kakaviatos N. Relation of sodium balance to arterial pressure during drug-induced saluresis. Circulation 1968; 37: 175-83. 6. Hollander W, Chobanian A, Wilkins RW. Relationship between diuretic and antihypertensive effects of chlorothiazide and mercurial diuretics. Circulation 1959; 19: 827-38. 7. Lauwers P, Conway J. Effect of long-term treatment with chlorothiazide on body fluids, serum electrolytes, and exchangeable sodium in hypertensive patients. J Lab Clin Med 1960; 56: 401-08. 8. Gifford RW, Mattox VR, Orvis AL, Sones DA, Rosevear JW. Effect of thiazide diuretics on plasma-volume, body electrolytes, and excretion of aldosterone in hypertension. Circulation 1961; 24: 1197-205. 9. Bourgoignie JJ, Catanzaro FJ, Perry HM. Renin-angiotensin-aldosterone system during chronic thiazide therapy of benign hypertension. Circulation 1968; 37: 27-35. 10. Beavers WR. Rat aorta and muscle electrolytes following chlorothiazide administration. Fed Proc 1960; 19: 99. 11. Shah S, Khatri I, Freis E. Mechanism of antihypertensive effect of thiazide diuretics. Am Heart J 1978; 95: 611-18. 12. Chalmers J, Tiller D, Horvath J, Bune A. Effects of timolol and hydrochlorothiazide on blood-pressure and plasma renin activity. Lancet 1976; ii: 328-31. 13. Hansen J. Hydrochlorothiazide in the treatment of hypertension. Acta Med
Scand 1968; 183: 317-21.
"The size of the pool from which we rescue our patients has the greatest impact on our results. It is in the unglamorous, undramatic, and frequently contentious area of prevention that the surgeon who makes part of his living from trauma might well return something to society ... improving safety for the local high-school football team, preaching on the dusty roads that lead to school groups or on the gustatory roads to Rotary luncheons, participating in local television shows on the value of helmets to cyclists, collecting and studying relevant data, or educating uninformed congressmen."-ALEXANDER J. WALK. In praise of surgical hedgehogs; trauma and the compleat surgeon. Bull Am Coll Surgeons 1979; 64: no.
10,4.
DIHYDROPTERIDINE REDUCTASE DEFICIENCY DIAGNOSIS BY ASSAYS ON PERIPHERAL BLOOD-CELLS RICHARD G. H. COTTON FRANK A. FIRGAIRA DAVID M. DANKS Genetics Research Unit, Royal Children’s Hospital Research Foundation and Department of Pœdiatrics, University of Melbourne, Australia
Human
peripheral lymphocytes, granulocytes, and platelets contain the enzyme dihydropteridine reductase. A simple procedure and assay for diagnosing dihydropteridine reductase on peripheral blood is described. In twelve controls the level of enzyme activity in a lymphocyte-platelet preparation was 29.5±4.8 (SD) nmol NADH oxidised/min/mg protein. Two heterozygotes for dihydropteridine reductase deficiency had activities of 6·4 and 10·3 nmol NADH oxidised/min/mg protein, respectively. Summary
Introduction DURING the past 5 years there has been rapid progress in the understanding of why progressive cerebral degeneration occurs in some patients with phenylketonuria despite early dietary treatment.l Clinical recognition of this new variant of phenylketonuria led to the demonstration of tetrahydrobiopterin (BH4) deficiency and consequent deficiency of cerebral production of neurotransmitters derived from tyrosine and tryptophan. The administration of these neurotransmitters, together with the usual dietary restrictions, was beneficial. 2.3 Claims that serum phenylalanine could be controlled by BH4 administration4 have subsequently been confirmed,5.6 and these cases can be treated with BH4 and neurotransmitters without dietary restriction. 6.7 These patients can be diagnosed by a single test-dose of BH4 (2 mg/kg orally) given to all babies with a positive Guthrie test before the phenylalanine restricted diet is started. Serum phenylalanine measured before and at 2 and 6 h after the dose8 falls to normal levels in patients with BH4 deficiency, whereas in those with classical phenylketonuria due to phenylalanine hydroxylase deficiency there is no change in serum phenylalanine.6.8 BH4 deficiency can be due to dihydropteridine reductase (DHPR) deficiency or be caused by a defect in denovo synthesis of BH4’ Until now DHPR deficiency has been proven by assay in liver biopsy specimens/’lO in cultivated fibroblasts,9.11 or in continuous lymphoid cell cultures transformed by Epstein-Barr virus." Support for diagnosis of a defect in BH4 synthesis has come from measurements of pterin precursors in blood and urine and from response of serum phenylalanine to administration of various precursors to BH44.66 In a baby shown to have BH4-responsive phenylketonuria the delay involved in measuring DHPR in cultivated cells is unacceptable, and liver biopsy has been necessary because DHPR had not been found in normal
peripheral lymphocytes or leucocytes.9 We describe here the application of an optimised ;assay" (utilising saturating concentrations of substrates and a higher [37°C] assay temperature) which shows that peripheral blood lymphocytes,neutrophils, and platelets have DHPR activity at levels useful for diag-
1261
nostic purposes. Activity is also described in red bloodcells. Genetic deficiency can be shown by tests on peripheral blood samples.
Methods
Separation of Peripheral Blood-cells Heparinised blood was separated by the discontinuous ’Ficoll-Isopaque’ density gradient technique of B0yum into two fractions: (A) ficoll-isopaque/plasma interface cells (lymphocytes, monocytes, and platelets), and (B) sedimented cells (granulocytes and erythrocytes). The cells found at the interface region (A) and the intermediate layer down to 2 mm from the erythrocyte meniscus were collected. This lymphocyte-platelet preparation was diluted and mixed with one volume of 0-15 mol/1 phosphate buffered saline (PBS), pH 7.4, and centrifuged at 5000 g for 30 min at 4°C. The cells were resuspended in an approximate fourfold volume of 0 -15 mol/1 KCI, freeze-thawed three times, and homogenised. The homogenate was then centrifuged at 12 000 g for 30 min at 4°C to pellet membranes. The supernatant was removed and assayed for DHPR activity.
Preparation of Purified Peripheral Blood-cells 50 ml of blood was obtained from two normal donors, and four major cell-groups were separated from the ficoll-isopaque
gradient. Lymphocytes were separated from the ficoll-isopaque/ plasma interface (A) by centrifugation at 700 g for 5 min and washed three times with PBS. The platelet-rich supernatant from the first 700 g lymphocyte preparation was collected. This was centrifuged twice more at 700 g to sediment contaminating red and white cells and then at 5000 g for 30 min to pellet the platelets. Granulocytes were separated from the erythrocytes by mixing of the ficoll-isopaque sedimented cells (B) with 2 volumes of PBS and 1 volume of 6% dextran T500 (Pharmacia) in PBS. The erythrocytes were allowed to sediment for 1 h after which the granulocyte-rich supernatant was collected and centrifuged at 700 g for 5 min. Contaminating erythrocytes in the pellet were lysed in 2 ml of distilled water for 1 min, and the granulocytes were then washed in PBS. This procedure was carried out three times. The erythrocytes sedimented in the granulocyte isolation step were washed three times with PBS. Total and differential cell-counts were done on each of the isolated cell suspensions to check purity. Extracts of the cells were prepared as described above and assayed for DHPR acti-
vity. DHPR
Assay
The modified assay for determination of DHPR activity in human cell-lines described recently" was used in this study. All assays were done in at least triplicate and at two concentrations of enzyme extract. Values shown in the tables are the averages of such multiple estimations.
Polyacrylamide Gel Electrophoresis Two non-denaturing gel disc gel electrophoresis
(1)
were used: done at 40C with 7.5% (w/v) to the method of Ornstein and
electrophoresis systems was
polyacrylamide gels according Davis,13 and (2) gradient gel electrophoresis (2.5% to 27% [w/v] polyacrylamide) was done as previously described.’a Staining for DHPR activity was done for 5 min with disc gels and 30 min for gradient gels by the procedure described by Cotton and Jennings.15 Protein was stained with Coomassie blue G250 and de-stained in 7% (v/v) acetic acid.
Results DHPR Levels in Lymphocyte-platelet Preparations Fraction (A) ficoll-isopaque-separated cells from a group of normal adult volunteers (aged 20-50 years) of both sexes were used to determine normal DHPR activity. Cell-counts and differentials showed that these cells were mainly lymphocytes and platelets (in 1/80 ratio). DHPR activity in these cells is shown in table i. In a blind experiment two blood samples obtained from the parents of a baby who died with DHPR deficiency", 16 were included with normal donor samples. The parents’ samples were identified correctly and the levels (% of mean) of activity were similar to those established previously in their cultured skin fibroblasts and continuous lymphoid cells (table I). DHPR activity was also measured in a group of
children being treated at this hospital (table I); no difference in DHPR activity could be observed between sexes and no trend with age was apparent in this or the adult group of controls. Investigation of sample handling showed that activity is retained in blood samples for 24 h at room temperature ; however, there is some erythrocyte contamination and a lowered platelet yield in the lymphocyte-platelet population obtained from such samples. The lymphocyte-platelet pellet may be stored frozen at —20°C; DHPR determination on two such samples 7 days later showed normal levels of activity. DHPR in Purified
Blood-cells
The DHPR activity in isolated lymphocytes, platelets, and granulocytes from two normal donors is presented in table n. The level of activity in the lymphocyte preparation is similar to that seen in human continuous lym-
phoid cells. 11I Attempts to assay erythrocyte extracts gave inconclusive findings since a very high level of non-specific oxidation of NADH occurred without the pterin substrate. No activity could be detected in serum from six normal controls.
Inhibition ofEnzymeActivity in Lymphocyte-platelet Preparations Extracts of these cells were incubated with known inhibitors of DHPR for 5 min at 37°C. Both 0-1 mmol/1 p-chloromercuribenzoic acid and 0-01 mmol/1 HgCl2 produced complete inhibition of activity. Preincubation with 0-1mmol/1 NADH for 10 min protected against this inhibition. These findings are in agreement with those observed by Cheema et al. 17 with sheep liver DHPR. The DHPR activity in these extracts depended on the presence of all the substrate and co-factor components in the assay reaction mixture.
Electrophoresis of DHPR from Human Blood-cells Electrophoresis in non-denaturing gels was done on samples of the cell extracts produced in this study and compared with purified human-liver DHPR.15 The gels were stained in situ for DHPR activity. Disc gels (separation according to charge) are seen in the accompanying figure. All extracts showed a prominent purple band
1262 TABLE I-DHPR ACTIVITY
(nmol NADH oxidised/mm/mg) IN
LYMPHOCYTE-PLATELET PREPARATIONS OF NORMAL CONTROLS AND PARENTS OF A CHILD WITH DHPR DEFICIENCY
I
I
I
I
iI
* Parents of patient who died of DHPR deficiency.3.16 Nos. in parentheses show activity as a percentage of the mean for that all type.
t Previously published.’1 t Samples of peripheral blood (3-5 ml) were obtained from eleven children (aged 4 mo - 8 yr age range; five females and six males) who had unrelated diseases.
which migrated with the same mobility as the purified human-liver enzyme. The gradient gel system (separation according to molecular size), showed a similar band of activity co-migrating with human liver DHPR. It is interesting to note that an erythrocyte extract gave a small activity band in both systems. Our finding of DHPR activity in mouse cell-lines derived from erythroblastomas (unpublished observation) suggests that this enzyme may even be found in erythrocytes. Discussion We have previously demonstrated the presence of DHPR activity in cultured continuous lymphoid cells," and in this study show that it is also present in peripheral white’blood-cells. It is difficult to be certain why Kaufman et al.9 and Abelson et al. 18 failed to detect DHPR activity in peripheral blood lymphocytes and leucocytes. We assume that differences in our assay procedures have led to our apparently conflicting results. Abelson et al.18 reported the presence of DHPR with a specific activity of 3 nmol NADH oxidised/min/mg protein in human platelets; this figure is only 11% of that observed in our study, which means that the assay procedure employed in their studies was less sensitive TABLE II-DHPR ACTIVITY IN ISOLATED BLOOD CELLS
I
*
Values are mean =SD in nmol NADH oxidised/min/mg; nos. in parentheses indicate no. of assays done. Identical activity was seen on cell extracts stored frozen and thawed 1 week later. t Non-specific oxidation of NADH precluded use use of this assay on
erythrocyte extracts.
Disc gel
electrophoresis of extracts from peripheral blood-cell preparations. (a’) and (a) represent purified human liver DHPR, 24 fLg and 0’2 {jLg, respectively; (b) lymphocyte extract 194 p.g; (c) platelet extract 248 p.g; (d) granulocyte extract 230 jig; (e) erythrocyte extract 400 jig, (f) and (f’) lymphocyte-platelet preparation (table t) 210 }ig. Samples (a’) and (f’) were stained for protein. Samples (a-f) were stained for DHPR activity. The faster migrating band in the erythrocyte extract (e) is haemoglobin.
possible explanation for their failure to detect actiin vity peripheral blood cell leucocytes. We believe that the activity shown in these peripheral blood leucocytes is truly due to DHPR because: 1. The enzyme activity was destroyed by appropriate inhibitors and by exclusion of components of the reaction mixture. 2. Heterozygotes for DHPR deficiency showed the expected result, these studies being done blind by the use of coded samples. 3. The level of residual activity observed in cells from these two heterozygotes was very similar to that found previously in their fibroblasts and continuous lymphoid cells. 4. The presence of DHPR has also been shown by a dif ferent approach consisting of gel electrophoresis followed by specific staining for enzyme activity. The band of activity moved to the same position as the purified enzyme from human liver on two different gel systems, which separated proteins according to charge and to molecular size, respectively. In designing a simple procedure for determination of the DHPR activity in a peripheral blood sample, we chose to use the lymphocyte-platelet preparation obtained by ficoll-isopaque separation. This technique is routinely available in most hospital laboratories and gives a good yield of DHPR-containing cells for a given
- a
1263
sample of blood. The difference in DHPR activity between lymphocytes and platelets is not great enough for variation in the relative numbers of these cells to disturb the result, at least for diagnosis of affected homozygotes (zero activity expected). Our results show that heterozygotes can also be identified. A cell-count on the preparation is suggested to check the lymphocyte/platelet ratio and to assist in the interpretation. The assay offered here is quick (1 day for cell preparation and enzyme determination) and simple. Moreover, only a small sample of peripheral blood is required, which eliminates the need for liver biopsies or the long delay incurred in waiting for cultured cells to grow. Activity is retained in heparinised blood samples for 24 h at room temperature or in cell pellets for more than 1 week at -20°C. Transport of samples to a specialist laboratory is therefore feasible. We thank Dr G. Tauro and Dr C. Hosking and their staff for their This work was supported partly by the National Health and Medical Research Council of Australia. F. A. F. was the recipient of a Commonwealth postgraduate research award.
help.
Requests for reprints should be addressed to D.M.D., Genetics Research Unit, Royal Children’s Hospital Research Foundation, Parkville, Victoria 3052, Australia.
REFERENCES
Clayton BE, Wolff OH. New variant of phenylketonuria with progressive neurological illness unresponsive to phenylalanine restriction.
1. Smith I,
Lancet 1975; i: 1108-11. 2. Bartholomé K, Byrd DJ. L-dopa and 5-hydroxytryptophan therapy in phenylketonuria with normal phenylalanine hydroxylase. Lancet 1975; ii: 1042. 3. Danks DM, Bartholomè K, Clayton BE, et al. Malign ant hyperphenylalaninæmia—current status (June, 1977). J Inher Metab Dis 1978; 1: 49-53. 4. Danks DM, Cotton RGH, Schlesinger P. Variant forms of phenylketonuria. Lancet 1976; i: 1236. 5. Schaub J, Daumling S, Curtius H-Ch, et al. Tetrahydrobiopterin therapy of atypical phenylketonuria due to defective dihydrobiopterin. Arch Dis Child 1978; 53: 674-76. 6. Curtis H-Ch, Niederwieser A, Viscontini M, et al. Atypical phenylketonuria due to tetrahydrobiopterin deficiency. Diagnosis and treatment with tetrahydrobiopterin, dihydrobiopterin and sepiapterin. Clin Chim Acta 1979; 93: 251-62. 7. Niederwieser A, Curtius H-Ch, Bettoni O, et al. Atypical phenylketonuria caused by 7,8-dihydrobiopterin synthetase deficiency. Lancet 1979; i: 131-33. 8. Danks DM, Cotton RGH, Schlesinger P. Diagnosis of malignant hyperphenylalaninæmia. Arch Dis Child 1979; 54: 329-30. 9. Kaufman S, Holtzman NA, Milstein S, Butler IJ, Krumholz A. Phenylketonuria due to a deficiency of dihydropteridine reductase. N Engl J Med
1975, 293: 785-90. 10. Brewster TG, Moskowitz MA, Kaufman S, Breslow JL, Sheldon M, Israel FA. Dihydropteridine reductase deficiency associated with severe neurologic disease and mild hyperphenylalaninæmia. Pediatrics 1979; 63: 94-99. 11. Firgaira FA, Cotton RGH, Danks DM. Human dihydropteridine reductase. A method for the measurement of activity in cultured cells, and its application to malignant hyperphenylalaninemia. Clin Chim Acta 1979; 95: 47-59. 12. Bøyum A. Separation of leucocytes from blood and bone marrow. Scand J Clin Lab Invest 1968; 21: (suppl 97). 13. Ornstein L, Davis BJ. Disc electrophoresis, I and II. Distilation Products Industries, Rochester, 1962. 14. Cotton RGH, Jennings I. Affinity chromatography of phenylalanine hydroxylase. The structure of a ptendine adsorbent. Eur J Biochem 1978; 85: 357-63. 15. Cotton RGH, Jennings I. A napthoquinone adsorbent for affinity chromatography of human dihydropteridine reductase. Eur J Biochem 1978; 83: 319-24. 16. Danks DM, Schlesinger P, Firgaira F, et al. Malignant hyperphenylalaninemia—clinical features, biochemical findings and experience with administration of biopterins. Pediatr Res 1979; 13: 1150-55. 17. Cheema S, Soldin SJ, Knapp A, Hofmann T, Scrimgeour KG. Properties or purified quinonoid dihydropterin reductase. Can J Biochem 1973; 51: 1229-39. 18. Abelson TH, Gorka C, Beardsley PG. Identification of dihydropteridine reductase in human platelets. Blood 1979; 53: 116-21.
DETECTION OF VIRUS-ASSOCIATED ANTIGEN IN SERUM AND LIVER OF PATIENTS WITH NON-A NON-B HEPATITIS* L. VITVITSKI A. M. PRINCE
C. TREPO B. BROTMAN
INSERM U 45 et Centre d’Epidémiologie CNRS LP 005440, Hôpital Edouard Herriot, Lyons, France; Virology Laboratory, Lindsley F. Kimball Institute, New York Blood Center, New York, U.S.A.; and Vilab II, Liberian Institute ofBiomedical Research, Robertsfield, Liberia
Summary
In a search for serological markers of non-A non-B (NANB) hepatitis, sera
from
repeatedly transfused and convalescent patients assayed by immunodiffusion against sera from 12 patients with early acute NANB hepatitis. A new antigen/antibody system distinct from HBsAg was demonstrated in 8 cases. To assess the specificity of the test, serial sera from 17 patients with acute hepatitis of known ætiology (10 due to hepatitis-B virus, 4 to hepatitis-A virus, 3 to drugs) were tested twice a month, together with sera from 14 NANB patients obtained during a prospective post-transfusion study. NANB antigen (Ag) was detected in at least one sample from 12 of the 14 NANB patients (86%) but in none of the other groups. NANB Ag appeared after or just before elevation of transaminase levels and was cleared before they fell to normal. 4 of 5 patients who showed seroconversion to NANB antibody (Ab) had transient hepatitis. In were
contrast, the alanine aminotransferase value returned to normal in only 1 of the 5 with persistent NANB antigenæmia during 6 months’ follow-up. NANB Ag was also demonstrated by immunodiffusion in liver extracts from patients with chronic NANB hepatitis with antigenæmia. Fluorescein-isothiocyanate-labelled gammaglobulins with strong NANB Ab activity revealed specific nuclear fluorescence in foci of hepatocytes on cryostat sections of these livers but in none of 6 control human livers. The results suggest that the antigen and antibody are specifically linked to NANB hepatitis of long incubation period.
Introduction THE development of sensitive serological assays for hepatitis A and B has led to the recognition of a new category of viral hepatitis known as non-A non-B (NANB). Successful transmission of the disease to chimpanzees confirmed its infectious nature.’-5 More than one virus may be involved. 6.7 NANB hepatitis may account for up to 90% of all cases of post-transfusion hepatitis8.9 as well as for a significant proportion of spor-
adic
cases.
The causative viruses have
not
been identi-
fied, although various particles have been implicated.’O-" The finding that NANB infections are characterised by long-lasting virxmia and a tendency to progress to chronic hepatitis encouraged a search for possible circulating antigens which could be used as markers of NANB viruses. Shirachi et al.,13 using immunodiffusion, and Prince et aI.,5 using radioimmunoassay, have reported the existence of such an antigen/antibody sys*This paper was presented in part at the International Symposium on Hepatitis, Munich, April 5-7, 1979.