1140 DIHYDROPTERIDINE REDUCTASE ACTIVITY WITH AND WITHOUT PHENYLALANINE
(2 mmol/1)
BLOOD CARULOPLASMIN IN SERUM AND DRIED BLOOD SPOTS FROM NORMAL
SUBJECTS OR
*
AND PATIENTS WITH
WILSON’S
DISEASE
MENKES’ KINKY-HAIR DISEASE
Enzyme activity nmol N.A.D.H. oxidised/min/mg protein at 20 °C, as (and range). The components of the standard assay system
median were
was
essentially
as
described
previously.3.4
The
inter-assay
variation
8% and the day-to-day variation was 5%.
liver extract restores the activity, while none of the other three components substitutes for the hydroxylase.2 Schlesinger et al. reported that dihydropteridine reductase activity of P.K.U. fibroblasts is inhibited when the reductase is assayed in the presence of 2 mmol;1 phenylalanine.2 This finding, if correct, would open the way for intrauterine diagnosis and could explain the mental retardation in P.K.U.2 We have assayed dihydropteridine reductase activity in the presence and absence of 2 mmol/1 phenylalanine in fibroblast extracts of sixteen normal children with no family history of P.K.u. and of sixteen children with classical P.K.U. Phenylalanine had no effect on dihydropteridine-reductase activity in fibroblasts from either normal or P.K.U. children (see table). The activities in normal and P.K.U. fibroblasts accord with those reported previously.4 Neither a tenfold reduction in the concentration of 6,7-dimethyltetrahydropterin nor replacement of the synthetic cofactor with the natural cofactor, tetrahydrobiopterin (gift of Hoffmann-La Roche Inc.), resulted in any significant stimulation or inhibition when phenylalanine was added to the assay system. Nor could we confirm a stimulation or inhibition of dihydropteridine reductase by phenylalaninewhether the enzyme was assayed at 3 7°C or in an assay system containing half the concentration of the components mentioned above. Thus we cannot confirm the suggestion of Schlesinger et al. that phenylalanine can regulate the activity of dihydropteridine reductase, the principal enzyme responsible for keeping the hydroxylation cofactor biopterin in its fully reduced or active form. The high levels of phenylalanine seen in the tissues of untreated P.K.U. patients, cannot therefore play a role in regulating the synthesis of neurotransmitters by inhibiting dihydropteridine reductase in neural tissues, nor do they explain mental retardation in P.K.U. It is also not possible to distinguish P.K.U. phenotypes in fibroblasts on this basis. John F. Kennedy Institute, DK-2600 Glostrup, Denmark
F. GÜTTLER
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20014, USA.
S. KAUFMAN S. MILSTIEN
NEW SCREENING METHOD FOR WILSON’S DISEASE AND MENKES’ KINKY-HAIR DISEASE
SIR,-Wilson’s diseases and Menkes’ kinky-hair disease6 are characterised
by a relative or total absence of serum cmruloplasmin. Early diagnosis and treatment of these diseases which may prevent brain-damage and other symptoms is difficult because blood caeruloplasrnin in newborns and infants is lower than in adults. We describe here a simple and inexpensive 2. Schlesinger, P., Cotton, R. G. H., Danks, D. M. Lancet 1976, ii, 1245. 3. Craine, J. E., Hall, E. S., Kaufman, S. J. biol. Chem. 1972, 247, 6082. 4. Milstien, S., Holtzman, N. A., O’Flynn, M. E., Thomas, G. H., I. J., Kaufman, S. J. Pediat. 1976, 89, 763.
5. 6.
Butler,
Sternlib, I., Scheinberg, I. H. J. Am. med. Ass. 1964, 189, 748. Danks, D. M., Stevens, B. J., Campbell, P. E., Gillespie, J. M., WalkerSmith, J., Blomfield, J. Lancet, 1972, i, 1100.
*detected first by the dried blood spot technique. Values are mean+2 S.D.
method of measuring blood caeruloplasmin using a dried blood spot, which may be of value in screening for early diagnosis. Blood is spotted on to filterpaper and serum is obtained from a sample collected in a capillary tube. Both samples are stored at -20°C. Two 3 mm diameter discs are punched out from the filterpaper, one for testing and the other as a control. Each disc is placed in 50 1 distilled water in a 8-9 mm diameter test tube. After several hours at room temperature, 100 lil fresh 0-5% p-phenylenediamine followed by 250 fl1 of 0.1 mol/1 acetate buffer (pH 4.0)1 is added to each tube. 0.3% sodium azide (NaN3) is added to the control and both tubes are incubated at 37°C for 120 min. On cooling 100 fl1 0.3% NaN3 solution is added to the test solution. An immediate purplishred or brownish-red colour denotes a positive reaction and the change in optical density (A o.D.) is measured by spectrophotometry at a wavelength of 560 nm. When this procedure fails to distinguish clearly between the test and the control, cmruloplasmin can be measured immunologicauyg in the serum obtained from the capillary tube blood sample. In a trial of the dried-blood method, normal values of 4o.n. were established from data given by 94.7% full-term newborn infants tested, 98.0% 1-month-old infants, 100% 3-4 monthold infants and 100% adults with a normal serum ceeruloplasmin (determined immunologically) (see table). In the infants with abnormally low Ao.D., the relative or total absence of caeruloplasmin was confirmed by immunological means. It is thus possible that adequate screening for infants of this age may be provided by the dried blood spot method alone. Department of Pædiatrics Toho University School of Medicine,
Tokyo, Japan
TSUGUTOSHI AOKI MASAKO NAKAHASHI
DRUG ADDICTION
SIR,--Of the first interim report of the Treatment and Rehabilitation Working Group of the Advisory Council on the Misuse of Drugs you say (Oct. 22, p. 861) "the evidence for what the report calls ’a slowly worsening problem’ is not con7. Ravin, H. A. J. lab. clin. Med. 1961, 58, 161. 8. Fahey, J. L., McKelvey, E. M. J. Immun. 1965,
94, 84.
1141
vincing : the number of heroin addicts has risen by 37% (1426 to 1954) over the six years 1970 to 1975, inclusive, but the rate of climb seems to be slowing." These figures refer to addicts known to be receiving drugs as at Dec. 31, and this figure, at the end of 1975, was 1954. The Home Office news release of Oct. 7, 1976, on 1975 Statistics of the Misuse of Drugs in the United Kingdom, gives the following figures for narcotic drug addicts known to the Home Office in 1975, by new notifications and numbers no longer recorded as drug addicts.
Definition
No.
Addicts known to be receiving drugs at Jan. 1, 1975 1972 Notifications by medical practitioners of: New addicts during 1975 926 Former addicts renotified 532 Addicts no longer recorded as at Dec. 31, 1975: Died 69 Admitted to a penal institution 483 Admitted to other institution 1 No longer seeking treatment 923 Addicts known to be receiving drugs as at Dec. 31, 1975 1954
Cytogenetics Laboratory, Department of Pathology, Letterman Army Medical Center, Presidio of San Francisco, California 94129, U.S.A.
If you add the numbers of new addicts in 1975 (926) and the number of former addicts renotified (532) to the number known to be receiving narcotic drugs on Jan. 1 (1972) you will see that the total numbers of addicts seen throughout 1975 was 3430. Also, if you add the number who died (69), those admitted to a special institution (483), and those no longer seeking treatment (926), you will see that 1475 were no longer recorded as at Dec. 31,1975. I bring these figures to your notice as your excellent journal is widely read in the United States and it could be that the report is correct in its opinion that there is evidence of a slowly
worsening problem. Division of Resource Development, National Institute on Drug Abuse, Rockville, Maryland 20857, U.S.A.
RICHARD V. PHILLIPSON
TWO-MINUTE METHOD FOR BANDING HUMAN CHROMOSOMES
StR,-For the past 2 years I have been trying to achieve a Giemsa chromosome banding method that is reliable, simple, fast, and cheap. Such a method has been devised with the addition of acetone to the stain which reduces the staining-time
Part of metaphase
spread showing banding achieved
acetone/Giemsa method.
with
and produces more distinct bands. Starting with air-dried slides the whole procedure can be done at room temperature in under 2 min. Human leucocytes are cultured to obtain metaphases using 75 mmol/1 potassium chloride as the hypotonic solution for 4 min at 37°C and fixation with a freshly prepared mixture of glacial acetic acid and methanol (1/3) at least three times before transfer to clean, dry slides by blowing. The slide is then submerged horizontally in 0.25% trypsin (Difco) in physiological saline solution for 10-15 s and then passed through two rinses of saline. At this point the slide is stained horizontally for 1 min with Giemsa stain (Reagent Chemicals, Saugerties, N.Y.) diluted 1/4 with pH 6.8 buffer salts (Reagent Chemicals) plus the addition of 0.75% acetone. The slide is rinsed well in pH 6.8buffer salts, blotted dry on filterpaper, rinsed in xylol, and mounted in a neutral mounting medium. This method has resulted in consistent Giemsa bands for the past 8 months.
rapid
Commentary
from Westminster
From Our Parliamentary
The Next
RICHARD J. DERR
Correspondent
Stage on the Consultants’ Contract
AFTER negotiations which have lasted for most of the year, details of a proposed new contract for consultants have now been agreed with the Government and are expected to be finalised this week. The proposed contract, to cover all but exceptional cases, is for a mini-
of ten notional half-days of 3 Zhours a week, comprising eight sessions of scheduled work, one for administrative and committee work, and one for on-call commitment in recognition of the fact that a consultant continues to accept responsibility for his patients and his own department even when he is not officially at work. Extra sessions would then be offered by health authorities, who would take into account a consultant’s outside commitments to private practice. At the moment, full-. time consultants in the National Health Service work eleven sessions of 3t hours each a week and undertake not to do private practice. Part-time consultants work nine sessions and lose two-elevenths of their pay, but are allowed to do private work. The aim of the Department of Health and Social Security has been to increase the commitment of consultants to the National Health Service, although whether the proposed contract achieves this will be one of the main areas of dispute. The consultants’ negotiating team has been anxious to see that consultants get more money in order to make the job more attractive for juniors. Indeed, it has been largely with the younger generation of consultants and juniors in mind that the new contract has been drawn up. The aim of the consultants has been to negotiate a contract which identifies what they are being paid for and flexible enough to allow payment for additional work. But in doing this they did not want to be tied down to a tightly timed agreement. In working out the details of the new contract the two sides have faced a number of difficulties. mum
(2 mmol/1)
BLOOD CARULOPLASMIN IN SERUM AND DRIED BLOOD SPOTS FROM NORMAL
SUBJECTS OR
*
AND PATIENTS WITH
WILSON’S
DISEASE
MENKES’ KINKY-HAIR DISEASE
Enzyme activity nmol N.A.D.H. oxidised/min/mg protein at 20 °C, as (and range). The components of the standard assay system
median were
was
essentially
as
described
previously.3.4
The
inter-assay
variation
8% and the day-to-day variation was 5%.
liver extract restores the activity, while none of the other three components substitutes for the hydroxylase.2 Schlesinger et al. reported that dihydropteridine reductase activity of P.K.U. fibroblasts is inhibited when the reductase is assayed in the presence of 2 mmol;1 phenylalanine.2 This finding, if correct, would open the way for intrauterine diagnosis and could explain the mental retardation in P.K.U.2 We have assayed dihydropteridine reductase activity in the presence and absence of 2 mmol/1 phenylalanine in fibroblast extracts of sixteen normal children with no family history of P.K.u. and of sixteen children with classical P.K.U. Phenylalanine had no effect on dihydropteridine-reductase activity in fibroblasts from either normal or P.K.U. children (see table). The activities in normal and P.K.U. fibroblasts accord with those reported previously.4 Neither a tenfold reduction in the concentration of 6,7-dimethyltetrahydropterin nor replacement of the synthetic cofactor with the natural cofactor, tetrahydrobiopterin (gift of Hoffmann-La Roche Inc.), resulted in any significant stimulation or inhibition when phenylalanine was added to the assay system. Nor could we confirm a stimulation or inhibition of dihydropteridine reductase by phenylalaninewhether the enzyme was assayed at 3 7°C or in an assay system containing half the concentration of the components mentioned above. Thus we cannot confirm the suggestion of Schlesinger et al. that phenylalanine can regulate the activity of dihydropteridine reductase, the principal enzyme responsible for keeping the hydroxylation cofactor biopterin in its fully reduced or active form. The high levels of phenylalanine seen in the tissues of untreated P.K.U. patients, cannot therefore play a role in regulating the synthesis of neurotransmitters by inhibiting dihydropteridine reductase in neural tissues, nor do they explain mental retardation in P.K.U. It is also not possible to distinguish P.K.U. phenotypes in fibroblasts on this basis. John F. Kennedy Institute, DK-2600 Glostrup, Denmark
F. GÜTTLER
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20014, USA.
S. KAUFMAN S. MILSTIEN
NEW SCREENING METHOD FOR WILSON’S DISEASE AND MENKES’ KINKY-HAIR DISEASE
SIR,-Wilson’s diseases and Menkes’ kinky-hair disease6 are characterised
by a relative or total absence of serum cmruloplasmin. Early diagnosis and treatment of these diseases which may prevent brain-damage and other symptoms is difficult because blood caeruloplasrnin in newborns and infants is lower than in adults. We describe here a simple and inexpensive 2. Schlesinger, P., Cotton, R. G. H., Danks, D. M. Lancet 1976, ii, 1245. 3. Craine, J. E., Hall, E. S., Kaufman, S. J. biol. Chem. 1972, 247, 6082. 4. Milstien, S., Holtzman, N. A., O’Flynn, M. E., Thomas, G. H., I. J., Kaufman, S. J. Pediat. 1976, 89, 763.
5. 6.
Butler,
Sternlib, I., Scheinberg, I. H. J. Am. med. Ass. 1964, 189, 748. Danks, D. M., Stevens, B. J., Campbell, P. E., Gillespie, J. M., WalkerSmith, J., Blomfield, J. Lancet, 1972, i, 1100.
*detected first by the dried blood spot technique. Values are mean+2 S.D.
method of measuring blood caeruloplasmin using a dried blood spot, which may be of value in screening for early diagnosis. Blood is spotted on to filterpaper and serum is obtained from a sample collected in a capillary tube. Both samples are stored at -20°C. Two 3 mm diameter discs are punched out from the filterpaper, one for testing and the other as a control. Each disc is placed in 50 1 distilled water in a 8-9 mm diameter test tube. After several hours at room temperature, 100 lil fresh 0-5% p-phenylenediamine followed by 250 fl1 of 0.1 mol/1 acetate buffer (pH 4.0)1 is added to each tube. 0.3% sodium azide (NaN3) is added to the control and both tubes are incubated at 37°C for 120 min. On cooling 100 fl1 0.3% NaN3 solution is added to the test solution. An immediate purplishred or brownish-red colour denotes a positive reaction and the change in optical density (A o.D.) is measured by spectrophotometry at a wavelength of 560 nm. When this procedure fails to distinguish clearly between the test and the control, cmruloplasmin can be measured immunologicauyg in the serum obtained from the capillary tube blood sample. In a trial of the dried-blood method, normal values of 4o.n. were established from data given by 94.7% full-term newborn infants tested, 98.0% 1-month-old infants, 100% 3-4 monthold infants and 100% adults with a normal serum ceeruloplasmin (determined immunologically) (see table). In the infants with abnormally low Ao.D., the relative or total absence of caeruloplasmin was confirmed by immunological means. It is thus possible that adequate screening for infants of this age may be provided by the dried blood spot method alone. Department of Pædiatrics Toho University School of Medicine,
Tokyo, Japan
TSUGUTOSHI AOKI MASAKO NAKAHASHI
DRUG ADDICTION
SIR,--Of the first interim report of the Treatment and Rehabilitation Working Group of the Advisory Council on the Misuse of Drugs you say (Oct. 22, p. 861) "the evidence for what the report calls ’a slowly worsening problem’ is not con7. Ravin, H. A. J. lab. clin. Med. 1961, 58, 161. 8. Fahey, J. L., McKelvey, E. M. J. Immun. 1965,
94, 84.
1141
vincing : the number of heroin addicts has risen by 37% (1426 to 1954) over the six years 1970 to 1975, inclusive, but the rate of climb seems to be slowing." These figures refer to addicts known to be receiving drugs as at Dec. 31, and this figure, at the end of 1975, was 1954. The Home Office news release of Oct. 7, 1976, on 1975 Statistics of the Misuse of Drugs in the United Kingdom, gives the following figures for narcotic drug addicts known to the Home Office in 1975, by new notifications and numbers no longer recorded as drug addicts.
Definition
No.
Addicts known to be receiving drugs at Jan. 1, 1975 1972 Notifications by medical practitioners of: New addicts during 1975 926 Former addicts renotified 532 Addicts no longer recorded as at Dec. 31, 1975: Died 69 Admitted to a penal institution 483 Admitted to other institution 1 No longer seeking treatment 923 Addicts known to be receiving drugs as at Dec. 31, 1975 1954
Cytogenetics Laboratory, Department of Pathology, Letterman Army Medical Center, Presidio of San Francisco, California 94129, U.S.A.
If you add the numbers of new addicts in 1975 (926) and the number of former addicts renotified (532) to the number known to be receiving narcotic drugs on Jan. 1 (1972) you will see that the total numbers of addicts seen throughout 1975 was 3430. Also, if you add the number who died (69), those admitted to a special institution (483), and those no longer seeking treatment (926), you will see that 1475 were no longer recorded as at Dec. 31,1975. I bring these figures to your notice as your excellent journal is widely read in the United States and it could be that the report is correct in its opinion that there is evidence of a slowly
worsening problem. Division of Resource Development, National Institute on Drug Abuse, Rockville, Maryland 20857, U.S.A.
RICHARD V. PHILLIPSON
TWO-MINUTE METHOD FOR BANDING HUMAN CHROMOSOMES
StR,-For the past 2 years I have been trying to achieve a Giemsa chromosome banding method that is reliable, simple, fast, and cheap. Such a method has been devised with the addition of acetone to the stain which reduces the staining-time
Part of metaphase
spread showing banding achieved
acetone/Giemsa method.
with
and produces more distinct bands. Starting with air-dried slides the whole procedure can be done at room temperature in under 2 min. Human leucocytes are cultured to obtain metaphases using 75 mmol/1 potassium chloride as the hypotonic solution for 4 min at 37°C and fixation with a freshly prepared mixture of glacial acetic acid and methanol (1/3) at least three times before transfer to clean, dry slides by blowing. The slide is then submerged horizontally in 0.25% trypsin (Difco) in physiological saline solution for 10-15 s and then passed through two rinses of saline. At this point the slide is stained horizontally for 1 min with Giemsa stain (Reagent Chemicals, Saugerties, N.Y.) diluted 1/4 with pH 6.8 buffer salts (Reagent Chemicals) plus the addition of 0.75% acetone. The slide is rinsed well in pH 6.8buffer salts, blotted dry on filterpaper, rinsed in xylol, and mounted in a neutral mounting medium. This method has resulted in consistent Giemsa bands for the past 8 months.
rapid
Commentary
from Westminster
From Our Parliamentary
The Next
RICHARD J. DERR
Correspondent
Stage on the Consultants’ Contract
AFTER negotiations which have lasted for most of the year, details of a proposed new contract for consultants have now been agreed with the Government and are expected to be finalised this week. The proposed contract, to cover all but exceptional cases, is for a mini-
of ten notional half-days of 3 Zhours a week, comprising eight sessions of scheduled work, one for administrative and committee work, and one for on-call commitment in recognition of the fact that a consultant continues to accept responsibility for his patients and his own department even when he is not officially at work. Extra sessions would then be offered by health authorities, who would take into account a consultant’s outside commitments to private practice. At the moment, full-. time consultants in the National Health Service work eleven sessions of 3t hours each a week and undertake not to do private practice. Part-time consultants work nine sessions and lose two-elevenths of their pay, but are allowed to do private work. The aim of the Department of Health and Social Security has been to increase the commitment of consultants to the National Health Service, although whether the proposed contract achieves this will be one of the main areas of dispute. The consultants’ negotiating team has been anxious to see that consultants get more money in order to make the job more attractive for juniors. Indeed, it has been largely with the younger generation of consultants and juniors in mind that the new contract has been drawn up. The aim of the consultants has been to negotiate a contract which identifies what they are being paid for and flexible enough to allow payment for additional work. But in doing this they did not want to be tied down to a tightly timed agreement. In working out the details of the new contract the two sides have faced a number of difficulties. mum
