- Email: [email protected]
RENAL, HAEMODYNAMIC, AND HORMONAL EFFECTS OF HUMAN ALPHA ATRIAL NATRIURETIC PEPTIDE IN HEALTHY VOLUNTEERS
545 and disruptive to the social life of the children and families. There is a danger that parents who are hostile to their children may use the diet as a means of punishment or that parents may attribute all alteration of behaviour to diet. Others, for complex emotional reasons, may invent a story of overactivity in a normal child and introduce a rigorous diet with no medical justification-a form of Munchhausen’s syndrome by proxy.21 Therefore the diet should be planned only for severely affected children and, whenever possible, a double-blind, controlled provocation study should be done to confirm the diagnosis of food intolerance. In several children the initial improvement produced by the diet was preceded by a deterioration in behaviour. This is difficult for the family to accept, but means that it is necessary to persist for 2-3 weeks to gauge the effects. Giving full helpings of possibly provoking food for as long as a week in the reintroduction period is important to exclude a reaction. Being on an acceptable diet did seem to make a remarkable difference to the lives of many of these families. Many of the children, however, still have considerable behaviour problems, and they and their families continue to require counselling and advice concerning management of the child’s behaviour. Nevertheless, of the 62 children who responded, 57 (92%) were continuing the diet when last seen, despite the effort and expense involved. The observation that some patients ceased to react to certain foods suggests that recovery from this form of food allergy can occur, as it may in others. Further work along these lines is clearly necessary for a variety of reasons. It would first be desirable to determine, by means of tighter double-blind design, to what degree the effectiveness of the diet is due to non-specific factors. If the specific effect apparently demonstrated in this study is confirmed, then further trials of treatment with more typically affected children need to be conducted. The mode of administration of the diet also needs to be simplified to make it more easily applicable. ’
We thank Dr Wilson, Dr D. Black, Mrs M. Lobascher, and others for permitting us to study their patients; Messrs Heinz and Kelloggs for generous supply of foods for the study; and Ms L. Maddern for help in the development of psychological tests. The work was supported by a grant from the Medical Research Council to J. F. S. and P. J. G.
Correspondence should be addressed to J. E., Haunersches Lmdwurmstrasse 4, 8000 Munich 2, West Germany.
Kinderspnal,
REFERENCES
Hyperkinetic syndrome. In: Rutter M, Hersov L, eds. Child psychiatry: modern approaches. Oxford. Blackwell, 1976: 524-55 2 Feingold BF Hyperkinesis and learning disabilities linked to artificial food flavours and colors Am J Nurs 1976, 75: 797-803. 3 Conners CK, Goyette CH, Southwick DA, Lees JM, Andrulonis P Food additives and hyperkinesis a controlled double blind experiment Pediatrics 1976; 58: 154-66. 4 Harley JP, Ray RS, Tomasi L, et al. Hyperkinesis and food additives, testing the Feingold hypothesis. Pediatrics 1970; 61: 818-28. 5 Swanson JM, Kinsbourne M Food dyes impair performance of hyperactive children on a laboratory learning test. Science 1980; 207: 1485-87 6 Goyette CH, Conners CK, Petti TA, Curtis LE. Effects of artificial colors on hyperkinetic children a double blind challenge study. Psychopharmacol Bull 1978; 14: 39-40 7 Thorley G Pilot study to assess behavioural and cognitive effects of artificial food colours in a group of retarded children. Devl Med Child Neurol 1984; 26: 56-61 8 Weiss B, Williams JH, Margen S, et al. Behavioral response to artificial food colors. Science 1980, 207: 1487-89 9 Rowe AH Food allergy: its manifestations, diagnosis and treatment. Philadelphia: Lea and Febiger, 1931. 10 Randolph TG Allergy as a causative factor in fatigue, irritability and behavior problems in children. J Pediat 1947; 31: 560-72. Egger J. Carter CM, Wilson J, Turner MW, Soothill JF. Is migraine food allergy? A double-blind controlled trial of oligoantigenic diet treatment. Lancet 1983; n: 865-69 River ML, Shaffer D, Sturge C. A guide to a multi-axial classification scheme for psychiatric disorders in childhood and adolescence London: Institute of Psychiatry, 1975 13 Conners CK Food additives and hyperactive children New York: Plenum, 1980. I Cantwell D
RENAL, HAEMODYNAMIC, AND HORMONAL EFFECTS OF HUMAN ALPHA ATRIAL NATRIURETIC PEPTIDE IN HEALTHY
VOLUNTEERS A. M. RICHARDS H. IKRAM T. G. YANDLE
M. G. NICHOLLS M. W. I. WEBSTER E. A. ESPINER
Departments of Endocrinology and Cardiology, Hospital, Christchurch, New Zealand
Princess Margaret
The effects of atrial natriuretic peptide (ANP) were investigated in six healthy male volunteers taking a constant diet (120 mmol sodium and 60 mmol potassium daily). They were given an intravenous bolus of 100 µg human &agr;-ANP on one day or placebo on another day 1-3 weeks apart in a double-blind randomised study. After ANP, urinary sodium excretion increased fourfold, and urine volume, calcium, magnesium, and phosphorus excretion doubled within 30 min of the injection. ANP induced an immediate fall in arterial pressure, followed by a longer vasodepressor phase which exceeded the duration of the effect on electrolyte excretion. There were no significant changes in plasma renin activity, aldosterone, antidiuretic hormone, or noradrenaline when compared with
Summary
placebo. Introduction THE presence of specific granules in rat atrial myocytes was first reported in the 1950so The number of atrial granules is altered by changes influid and electrolyte balance.3 Extracts of both human and animal atria have potent diuretic and natriuretic effects in animals. 3-6 Atrial extracts relax vascular smooth muscle,7 inhibit the vasoconstrictor actions of angiotensin II and noradrenaline,8 and reduce aldosterone secretion.9 These observations suggest that atrial peptides help to regulate circulating volume, as well as vascular tone, and could be involved in the pathogenesis of heart failurelo and certain types of hypertension.I 1,12z Three polypeptide species have been isolated from human atria by Japanese workers, who have proposed the names a, P, and y human atrial natriuretic peptide (ANP).13,14 Each has natriuretic and vasorelaxing properties in experimental preparations, but their action in man is unknown. We have studied the effects of synthetic human a-ANP on urine excretion, arterial pressure, heart rate, and plasma hormones in healthy volunteers.
Subjects and Methods Synthetic human a-ANP, 28 aminoacids, was obtained from Bachem (Torrance, California). The purity of the peptide was confirmed by high-performance liquid chromatography. The peptide (2 mg) was dissolved in 4 rnl acid saline (0-9% sodium
CM, Egger J, Soothill JF. A dietary management of severe childhood migraine. Hum Nutr. Appl Nut (in press). Cairns E, Cammock T Development of a more reliable version of the matching familiar figures test Devl Psychol 1978; 14: 555-60. Porteus SD. The Porteus maze test manual. London Harrap, 1952 Wechsler intelligence scale for children New York: Psychological Corporation, 1974. Wechsler preschool and primary scale of intelligence. New York: Psychological
14. Carter 15 16. 17 18
Corporation, 1963. 19. Terman LM, Merrill MA Stanford-Bmet intelligence scale- Manual for the third revision form L-M. London Harrap, 1961 20. Hills M, Armitage P. The two-period cross-over clinical trial Br J Clin Pharmacol 1979; 8: 7 21 Warner JO, Hathaway MJ. Allergic form of Meadow’s syndrome (Munchausen by proxy). Arch Dis Child 1984; 59: 151.
’
546 chloride in 3 mmolll hydrochloric acid), made up to 20 ml with ’Haemaccel’ (Behring, West Germany), passed through a Gelman ’Acrodisc’ (0 -2 J.lm) filter, and stored as 1 ml volumes (each containing 100 pg a-ANP) at -70°C. Placebo consisted of acid saline in haemaccel, prepared and stored under the same conditions. The experimental protocol was approved by the hospital ethical committee. We studied six healthy male volunteers, hospital staff, aged 29-50 years (mean 34 - 5) who weighed 57-80 kg (mean 70 5 kg); none were taking medication. The subjects were studied on 2 days (1 day ANP, 1 day placebo) 1-3 weeks apart, on the 4th day of a caffeine-free diet with constant sodium (120 mmol/day) and potassium (60 mmol/day) content. While the subjects were taking the diet, they continued their normal hospital duties and slept at home. On the 4th day, they ate breakfast (0700 h), completed a 24 h urine collection (0800 h), were weighed, then entered the study room, which was kept at a temperature of 23, 5-25’ ooC. The non-dominant brachial artery was cannulated for continuous pressure and heart-rate recording by the Oxford technique, 15 and two peripheral venous lines were inserted-one for hormone sampling, one for injection ofANP or placebo. From 0830
h to 1330 h, the subjects remained seated in an easy chair apart from brief periods of standing to pass urine every 30 min. To ensure adequate urine flow, subjects drank distilled water equal in volume to the urine passed in the previous 30 min plus 50 ml. After four baseline urine collections ANP (100 Jig) or placebo was given intravenously over 1 min in a random, double-blind experimental design. Urine collections and arterial pressure recordings were continued until 1330 h. Venous samples were obtained before andat set times after ANP or placebo administration for measurements of plasma renin activity, I6 aldosterone,17 cortisol (enzyme-linked immunosorbent assay), catecholamines, 18 antidiuretic hormone, 19 and sodium and potassium (flame photometry). As each urine sample was passed, volume, pH, and osmolality (freezing-point depression) were measured immediately, and samples were taken for determination of sodium and potassium (flame photometry), calcium and magnesium (atomic absorption spectrophotometry), phosphorus (Gomorri’s colorimetric method), and creatinine
(Beckman analyser). Blood samples from the first and second study in each volunteer were assayed for hormones in the same run. Variability within assays ranged from 4 - 9% (coefficient of variation) for aldosterone to 9-0% for adrenaline. Results from ANP and placebo studies were compared by means of the t test for paired data. Data are given as mean±standard error.
Fig 1-Urinary volume and electrolyte excretion before and after administration (arrow) of placebo (open columns) or ANP (filled columns) to six normal subjects (absolute values).
*p<0’05,
fp<0’01,
:p<0’001
for differences between ANP and
placebo.
547
2-Heart rate and mean arterial pressure integrated from continuous arterial recordings for 30 min periods before and after administration (arrow) of placebo or ANP to six normal subjects.
Fig
Results The studies were carried out without complication and data collection was complete. Mean±SEM 24 h urinary excretion of sodium and potassium was 108±5 mmol and 53±3 mmol, respectively, the day before the subjects were given placebo and 112±8 mmol and 6l:t3 mmol the day before ANP (not significant). Mean body weight did not change between the studies.
Urine There were no significant differences in urine indices between the 2 experimental days for the 30 min collection periods just before administration of ANP or placebo (fig 1). The mean urinary volume for the 30 min after ANP injection was twice that after placebo (p<0 01, fig 1) and the volumes after ANP were somewhat higher than those after placebo for the rest of the study period. Four subjects were aware of bladder distension within 10 min of receiving ANP. Urinary sodium excretion in the 30 min after infusion was four times greater after ANP than after placebo and remained significantly greater for 1’ 5 h (fig 1). Potassium excretion was
Fig 3-Systolic and diastolic arterial pressure and heart
rate for successive 1 min intervals from 10 min before to 12 min after administration (arrow) of placebo or ANP to six normal subjects.
slightly greater after ANP than after placebo, but this difference was not significant (fig 1). The urinary output of calcium, magnesium, and phosphorus was increased by ANP administration (fig 1). For calcium and magnesium the higher excretion lasted 1 h, whereas phosphorus excretion remained significantly above placebo levels for 2 h (fig 1). Urine osmolality fell steadily on both experimental days, the change being greater after ANP. However, owing to wide variations between subjects, this difference was not significant for any 30 min collection period. Urinary creatinine excretion and pH were not significantly affected by ANP. Blood Pressure and Heart Rate There was an acute rise in arterial pressure and heart rate ’ just before administration of ANP or placebo as the subjects stood to pass urine (figs 2 and 3) This was a consistent feature
MEAN PLASMA HORMONE LEVELS BEFORE AND AFTER PLACEBO
PRA= plasma
rerun
activity; ADH=antidiuretic hormone. *p<0-05
for difference between ANP and
(P) AND
placebo.
ANP
548
of the blood-pressure and heart-rate response to standing and micturition. Immediately after administration of ANP, all six subjects showed an acute fall in arterial pressure and a rise in heart rate (figs 2 and 3). In four this was associated with a short-lived sensation of flushing, and in one facial flushing was apparent to observers for approximately 1 min. The difference between ANP and placebo in arterial pressure was significant by the second minute (mean fall below placebo level 5’ 5±2’ 4 and 5 - 2± 1’ 4 mmHg in systolic and diastolic pressure, respectively), and pressures after ANP remained slightly below those after placebo for most of the next 8 min (fig 3). The difference betweenANP and placebo in heart rate also reached significance in the second minute, the maximum difference being 13’ 5± 1 4 bpm during the fifth minute after injection (fig 3). Analysis of mean data recorded over 30 min periods showed that ANP administration was associated with an initial fall in arterial pressure and a later, sustained hypotensive effect (figs 2 and 3). Similarly, heart rate increased briefly after ANP and was higher than after placebo for the rest of the study period (fig 2). Hormones There were no significant differences in plasma renin activity, aldosterone, cortisol, noradrenaline, or antidiuretic hormone between the 2 study days before or afterinjection of ANP or placebo (see table). Plasma adrenaline levels appeared to fall after ANP, though the possibility that ANP in plasma interferes with the radio-enzymic techniques of measuring adrenaline needs to be tested. Plasma sodium and potassium levels were not altered by ANP.
the renin-angiotensin-aldosterone system for maintenance of volume homeostasis. Though differences in study conditions make comparisons difficult, some of the effects ofANP resemble those ofa small intravenous bolus of frusemide. Frusemide also causes maximum increases in urine volume and sodium excretion within 30 min of administration, 2I and in primates the effects on excretion of water, sodium, and calcium are similar.6 However, acute haemodynamic effects differ. Although very low doses of frusemide increase venous capacitance within 5 min of injection, 22 systemic vascular resistance rises and arterial pressure is unchanged23,24 or may actually rise. 21,22 Levels of antidiuretic hormone,25 plasma renin activity, and aldosterone26 increase after frusemide. A direct vasodilator action, combined with acute fluid volume depletion in the absence of a vasopressor hormone response, may explain the prolonged hypotensive effect we observed after ANP. Our findings show that ANP has definite natriuretic, diuretic, and vasodepressor effects in normal man. It may also inhibit secretion from the juxtaglomerular apparatus, adrenal medulla, and zona glomerulosa. However, the type and the concentration of the circulating peptide, or peptides, in man are presently unknown. Further trials and the development of an assay for the peptide in plasma will help to show whether ANP has a role in the normal regulation of arterial pressure and volume homeostasis or in the pathophysiology and therapy of various disease states, including congestive cardiac failure and some forms of hypertension.
against
We thank ward B3 nurses, special test sisters, biochemistry and steroid laboratories, Mrs Daphne Hinton, Mr Steve Joyce, Mrs Rose Richards, and Mrs Natalie Purdue. This study was supported by the National Heart Foundation of New Zealand and the Medical Research Council of New Zealand.
Discussion These studies show that a small dose of a-ANP has clear natriuretic and vasodepressor effects in normal man. The amount administered may have been somewhat less than the 100 g (33 nmol) nominated owing to losses incurred during preparation and injection of the peptide. After injection of ANP there was a rapid, vigorous response in urine volume and electrolyte excretion. The response patterns are similar to those reported in rats.14 Whether the urinary effects can be accounted for by direct tubular actions of A-ANP, small changes in glomerular filtration rate, or redistribution of intrarenal blood flow remains to be determined. The arterial pressure response to ANP appeared to be biphasic. Mean arterial pressure fell below placebo levels within 3 min of injection. After a brief return to placebo levels at 10 min a longer vasodepressor phase followed and blood pressure remained significantly depressed (compared with the placebo day) 175 min after injection. The mechanisms underlying the hypotensive effect of ANP are not defined by our study. It seems likely, however, that the immediate effect, associated with flushing in several subjects, reflects a direct vasodilator action of ANP. The reciprocal rise in heart rate is consistent with arterial baroreceptor activation, but we did not see any substantial rise in plasma catecholamines; in fact, plasma adrenaline actually fell after ANP. Apart from the fall in plasma adrenaline, there were no clear changes in hormone levels after ANP. However, some trends consistent with data from animal experiments were seen. Aldosterone levels tended to fall in the hour after ANP, despite the concurrent diuresis and fall in blood pressure. ANP inhibits both basal and stimulated aldosterone secretion from isolated rat9 and bovine adrenal cells.2° Such an action fits well with the concept of a humoral system balanced
Correspondence should be addressed to E. A. E., Department of Endocrinology, Princess Margaret Hospital, Cashmere Road, Christchurch 2, New Zealand. REFERENCES 1. Kisch B. Electron microscopy of the atrium of the heart: guinea pig. Exp Med Surg 1956; 14: 99-112. 2. Bompiani GD, Rouiller C, Hatt PY. Le tissu de conduction du coeur chez la rat. Etude au microscope electronique. Arch Mal Coeur 1959; 52: 1257. 3. de Bold AJ. Heart atria granularity effects of changes in water-electrolyte balance. Proc Soc Exp Biol Med 1979; 161: 508-11. 4. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic
response
to intravenous
injection of atrial
myocardial extract in rats. Life Sci 1981,
28: 89-94. 5. Borenstein
atrial
HB, Cupples WA, Sonnenberg H, Veress AT. The effect of a natriutetic
extract on
Physiol 1983;
renal
haemodynamics and urinary excretion in anaesthetized rats. J
334: 133-40.
MN, Gilmore JP. Natriuretic activity of human and monkey atria. Circ Res 1983; 53: 420-23. 7. Currie MG, Geller DM, Cole BR, et al. Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria. Science 1983; 221: 71-73. 8. Kleinert HD, Maack T, Atlas SA, Januszewicz A, Sealey JE, Laragh JH. Atrial
6. Nemeh
9. 10.
11.
12.
13. 14.
15. 16.
natriuretic factor inhibits angiotensin norepinephrine, and potassium-induced vascular contractility. Hypertension 1984; 6 (suppl 1) I-143-47. Atarashi K, Mulrow P, Franco-Saenz R, Snajdar R, Rapp J. Inhibition of aldosterone production by an atrial extract. Science 1984; 224: 992-94. Chimoskey JE, Spielman WS, Brandt MA, Heidemann SR. Cardiac atria of BIO 14·6 hamsters are deficient in natriuretic factor. Science 1984; 223: 820-22. Hirata Y, Ganguli M, Tobian L, Iwai J. Dahl S rats have increased natriuretic factor in atria but are markedly hyporesponsive to it. Hypertension 1984; 6 (suppl 1). 1-148-55. Sonnenberg H, Milojevic S, Chong CK, Veress AT. Atrial natriuretic factor: reduced cardiac content in spontaneously hypertensive rats. Hypertension 1983; 5: 672-75 Oikawa S, Imai M, Ueno A, et al Cloning and sequence analysis of cDNA encoding a precursor for human atrial natriuretic polypeptide. Nature 1984; 309: 724-26. Kangawa K, Matsuo H Purification and complete ammo acid sequence of &agr;-human atrial natriuretic polypeptide (&agr;-hANP). Biochem Biophys Res Commun 1984, 118: 131-39 Millar-Craig MW, Hawes D, Whittington J. New system for recording ambulatory blood pressure in man. Med Biol Eng Comput 1978; 16: 727-31. Dunn PJ, Espiner EA. Outpatient screening tests for primary aldosteronism. Aust NZ J
Med 1976, 6: 131-35. S, Espiner EA, Nicholls MG, Yandle TG. A direct radioimmunoassay aldosterone in plasma. Clin Chem 1983; 29: 268-71.
17. Lun
for
549 had been discovered by screening in the newborn child of the first family. Increasing blood phenylalanine concentrations prompted dietary treatment from day 4 of life. The child has developed normally. One of the two children in the second family has PKU-also identified by the neonatal screening programme-and has been on a phenylalanine-restricted diet from day 9. This child is now 4 years old and has developed normally.
PRENATAL DIAGNOSIS OF CLASSIC PHENYLKETONURIA BY DNA ANALYSIS FLEMMING GÜTTLER SAVIO L. C. WOO
ALAN S. LIDSKY
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA; and John F. Kennedy Institute, Glostrup, Denmark
Howard Hughes Medical Institute,
.
Southern Blot Analysis
Prenatal diagnosis of classic
phenylketonuria (PKU) was performed in two at-risk families by means of a cloned human phenylalanine hydroxylase gene probe which was used to analyse DNA isolated from cultured amniotic fluid cells. The diagnoses of a PKU fetus in one family and a heterozygous fetus in another family were confirmed after birth. The prenatal diagnosis procedure by DNA analysis can be confidently applied to 90% of caucasian families with previously affected children. Summary
Introduction PHENYLKETONURIA
(PKU) is an inborn deficiency of the phenylalanine hydroxylase (PAH) that converts phenylalanine to tyrosine. I-3 The deficiency results in raised serum phenylalanine concentrations and mental retardation4 if the child does not receive long-term treatment with a lowphenylalanine diet5 starting within the first weeks of life.6 Mass screening of blood phenylalanine in neonates is performed in many countries. PKU is transmitted as an autosomal recessive trait,3 and it occurs in approximately 1 in 10 000 white neonates. Since the enzyme is liver-specific and is not expressed in fibroblasts, prenatal diagnosis by enzyme analysis of amniocytes is not possible. We have developed a molecular method for prenatal diagnosis of PKU by DNA analysis. We have isolated a human phenylalanine hydroxylase cDNA clone that detects the corresponding gene in the human genome.’Because several restriction endonucleases produce polymorphic patterns in the phenylalanine hydroxylase locus, the gene probe can be used to analyse PKU families by restriction fragment length polymorphism. 7,8 Since the restriction fragment patterns and the disease state were tightly linked in the families analysed, analysis of polymorphisms can theoretically lead to prenatal diagnosis of PKU in families which already have children with PKU. 7,1 We report the first two instances in which PKU was diagnosed prenatally by determining the segregation of the mutant PAH genes identified by the polymorphic restriction fragments. enzyme
Patients and Methods Patients Two families who already had one PKU child wanted to know the of their current pregnancies to prepare for delivery. PKU
outcome
18
19
Peuler JD, Johnson GA. Simultaneous single isotope radioenzymatic assays of plasma norepinephrine, epinephrine and dopamine. Life Sci 1977; 21: 625-33. Sadler WA, Lynskey CP, Gilchrist NL, Espiner EA, Nicholls MG. A sensitive radioimmunoassay for measuring plasma anti-diuretic hormone in man. NZ Med J 1983, 96: 959-63.
20 Goodfriend
TL, Elliott ME, Atlas SA Actions of synthetic atrial natriuretic factor on glomerulosa. Life Sci 1984; 35: 1675-82. 21 Andreasen F, Hansen U, Husted SE, Mogensen CE, Pedersen EB. The influence of age on renal and extrarenal effects of frusemide. Br J Clin Pharmacol 1984; 18: 65-74 22 Johnston GD, Nicholls DP, Leahey WJ. The dose-response characteristics of the acute non-diuretic peripheral vascular effects of frusemide in normal subjects. Br J Clin Pharmacol 1984; 18: 75-81 bovine adrenal
of Genomic DNA
phPAH247 is a recombinant plasmid containing a full-length human cDNA for phenylalanine hydroxylase,9 -which has been used to identify additional polymorphisms in the human PAH locus. 10 The cDNA insert was subcloned into the Eco RI site ofpBR322 and isolated by digestion of phPAH247 DNA with Eco RI followed by preparative electrophoresis in low-melting point agarose gels. The isolated DNA fragment was radiolabelled to a specific activity of 2-3 x 10g dpm/g by nick translation."Total human genomic DNA was purified from cultured amniotic-fluid cells and from peripheral-blood leucocytes isolated as buffy coats from whole blood as previously described.5g of genomic DNA was digested to completion with various restriction enzymes in appropriate buffers. Gel electrophoresis and blotting of DNA fragments to nitrocellulose filters were performed by a modified procedure of Southern. I2 The nitrocellulose filters were incubated for 5 h at 42°C in a pre-hybridisation solution containing 45% formamide, 5 x Denhardt’s solution, 4xSSC (salt and sodium citrate), 0 1 mol/1 sodium phosphate, pH 6-5, 0-1% sodium pyrophosphate, and 250 laglml sheared, denatured, herring sperm DNA. Hybridisation was performed in the pre-hybridisation solution which also contained 10% dextran sulphate and 2x 106 cpm/ml 32P-labelled PAH cDNA probe at 42°C overnight. The nitrocellulose filters were washed twice in 2 x SSC, 0’ 5% sodium dodecyl sulphate (SDS) for 15 min at room temperature, and twice in 0-lx SSC, 0-1% SDS for 2 h at 680C followed by autoradiography at -70°C for 1-5 days. Serum phenylalanine concentration was determined fluorimetrically as previously described. 13
Results Both parents in family 1 are heterozygous for the Hind III polymorphism. They both have a PAH gene containing the 4’ 2 kb fragment and a second gene containing the 4’ 0 kb fragment (fig 1, lanes 1 and 2). Digestion of DNA isolated from leucocytes of the PKU child showed that he inherited the PAH genes containing the 4’ 0 kbHind III fragment from both parents (fig 1, lane 3). Therefore, in this family the PKU traits segregated with the PAH genes containing the 4’ 0 kb fragment. Since the phenylalanine hydroxylase genes in family 1 were informative for gene analysis, DNA was isolated from cultured amniotic-fluid cells of a fetus at risk and analysed by digestion with Hind III followed by hybridisation with PAH cDNA probe. The results revealed that the fetus was homozygous for the 4 - 0 kb fragment. Thus, the fetus had the same genotype as the PKU proband; after delivery PKU was confirmed in this infant. The infant
23. Mond 24.
H, Hunt D, Sloman G. Haemodynamic effects of frusemide in patients suspected of having acute myocardial infarction. Br Heart J 1974; 36: 44-53. Lal S, Murtagh JG, Pollock AM, Fletcher E, Binnion PF. Acute haemodynamic effects of frusemide in patients with normal and raised left atrial pressures. Br Heart J 1969; 31: 711-17.
25. De Lima J, Caillens H, Beaufils M, Adaillou R. Effects of frusemide-induced plasma volume reduction on plasma antidiuretic hormone in normal and hypertensive subjects Clin Nephrol 1981; 15: 246-51.
26. Rosenthal J, Boucher R, Nowaczynski W, Genest J. Acute changes in plasma volume, renin activity, and free aldosterone levels in healthy subjects following frusemide administration Canad J Physiol Pharmacol 1968; 46: 85-91.
We thank Dr Wilson, Dr D. Black, Mrs M. Lobascher, and others for permitting us to study their patients; Messrs Heinz and Kelloggs for generous supply of foods for the study; and Ms L. Maddern for help in the development of psychological tests. The work was supported by a grant from the Medical Research Council to J. F. S. and P. J. G.
Correspondence should be addressed to J. E., Haunersches Lmdwurmstrasse 4, 8000 Munich 2, West Germany.
Kinderspnal,
REFERENCES
Hyperkinetic syndrome. In: Rutter M, Hersov L, eds. Child psychiatry: modern approaches. Oxford. Blackwell, 1976: 524-55 2 Feingold BF Hyperkinesis and learning disabilities linked to artificial food flavours and colors Am J Nurs 1976, 75: 797-803. 3 Conners CK, Goyette CH, Southwick DA, Lees JM, Andrulonis P Food additives and hyperkinesis a controlled double blind experiment Pediatrics 1976; 58: 154-66. 4 Harley JP, Ray RS, Tomasi L, et al. Hyperkinesis and food additives, testing the Feingold hypothesis. Pediatrics 1970; 61: 818-28. 5 Swanson JM, Kinsbourne M Food dyes impair performance of hyperactive children on a laboratory learning test. Science 1980; 207: 1485-87 6 Goyette CH, Conners CK, Petti TA, Curtis LE. Effects of artificial colors on hyperkinetic children a double blind challenge study. Psychopharmacol Bull 1978; 14: 39-40 7 Thorley G Pilot study to assess behavioural and cognitive effects of artificial food colours in a group of retarded children. Devl Med Child Neurol 1984; 26: 56-61 8 Weiss B, Williams JH, Margen S, et al. Behavioral response to artificial food colors. Science 1980, 207: 1487-89 9 Rowe AH Food allergy: its manifestations, diagnosis and treatment. Philadelphia: Lea and Febiger, 1931. 10 Randolph TG Allergy as a causative factor in fatigue, irritability and behavior problems in children. J Pediat 1947; 31: 560-72. Egger J. Carter CM, Wilson J, Turner MW, Soothill JF. Is migraine food allergy? A double-blind controlled trial of oligoantigenic diet treatment. Lancet 1983; n: 865-69 River ML, Shaffer D, Sturge C. A guide to a multi-axial classification scheme for psychiatric disorders in childhood and adolescence London: Institute of Psychiatry, 1975 13 Conners CK Food additives and hyperactive children New York: Plenum, 1980. I Cantwell D
RENAL, HAEMODYNAMIC, AND HORMONAL EFFECTS OF HUMAN ALPHA ATRIAL NATRIURETIC PEPTIDE IN HEALTHY
VOLUNTEERS A. M. RICHARDS H. IKRAM T. G. YANDLE
M. G. NICHOLLS M. W. I. WEBSTER E. A. ESPINER
Departments of Endocrinology and Cardiology, Hospital, Christchurch, New Zealand
Princess Margaret
The effects of atrial natriuretic peptide (ANP) were investigated in six healthy male volunteers taking a constant diet (120 mmol sodium and 60 mmol potassium daily). They were given an intravenous bolus of 100 µg human &agr;-ANP on one day or placebo on another day 1-3 weeks apart in a double-blind randomised study. After ANP, urinary sodium excretion increased fourfold, and urine volume, calcium, magnesium, and phosphorus excretion doubled within 30 min of the injection. ANP induced an immediate fall in arterial pressure, followed by a longer vasodepressor phase which exceeded the duration of the effect on electrolyte excretion. There were no significant changes in plasma renin activity, aldosterone, antidiuretic hormone, or noradrenaline when compared with
Summary
placebo. Introduction THE presence of specific granules in rat atrial myocytes was first reported in the 1950so The number of atrial granules is altered by changes influid and electrolyte balance.3 Extracts of both human and animal atria have potent diuretic and natriuretic effects in animals. 3-6 Atrial extracts relax vascular smooth muscle,7 inhibit the vasoconstrictor actions of angiotensin II and noradrenaline,8 and reduce aldosterone secretion.9 These observations suggest that atrial peptides help to regulate circulating volume, as well as vascular tone, and could be involved in the pathogenesis of heart failurelo and certain types of hypertension.I 1,12z Three polypeptide species have been isolated from human atria by Japanese workers, who have proposed the names a, P, and y human atrial natriuretic peptide (ANP).13,14 Each has natriuretic and vasorelaxing properties in experimental preparations, but their action in man is unknown. We have studied the effects of synthetic human a-ANP on urine excretion, arterial pressure, heart rate, and plasma hormones in healthy volunteers.
Subjects and Methods Synthetic human a-ANP, 28 aminoacids, was obtained from Bachem (Torrance, California). The purity of the peptide was confirmed by high-performance liquid chromatography. The peptide (2 mg) was dissolved in 4 rnl acid saline (0-9% sodium
CM, Egger J, Soothill JF. A dietary management of severe childhood migraine. Hum Nutr. Appl Nut (in press). Cairns E, Cammock T Development of a more reliable version of the matching familiar figures test Devl Psychol 1978; 14: 555-60. Porteus SD. The Porteus maze test manual. London Harrap, 1952 Wechsler intelligence scale for children New York: Psychological Corporation, 1974. Wechsler preschool and primary scale of intelligence. New York: Psychological
14. Carter 15 16. 17 18
Corporation, 1963. 19. Terman LM, Merrill MA Stanford-Bmet intelligence scale- Manual for the third revision form L-M. London Harrap, 1961 20. Hills M, Armitage P. The two-period cross-over clinical trial Br J Clin Pharmacol 1979; 8: 7 21 Warner JO, Hathaway MJ. Allergic form of Meadow’s syndrome (Munchausen by proxy). Arch Dis Child 1984; 59: 151.
’
546 chloride in 3 mmolll hydrochloric acid), made up to 20 ml with ’Haemaccel’ (Behring, West Germany), passed through a Gelman ’Acrodisc’ (0 -2 J.lm) filter, and stored as 1 ml volumes (each containing 100 pg a-ANP) at -70°C. Placebo consisted of acid saline in haemaccel, prepared and stored under the same conditions. The experimental protocol was approved by the hospital ethical committee. We studied six healthy male volunteers, hospital staff, aged 29-50 years (mean 34 - 5) who weighed 57-80 kg (mean 70 5 kg); none were taking medication. The subjects were studied on 2 days (1 day ANP, 1 day placebo) 1-3 weeks apart, on the 4th day of a caffeine-free diet with constant sodium (120 mmol/day) and potassium (60 mmol/day) content. While the subjects were taking the diet, they continued their normal hospital duties and slept at home. On the 4th day, they ate breakfast (0700 h), completed a 24 h urine collection (0800 h), were weighed, then entered the study room, which was kept at a temperature of 23, 5-25’ ooC. The non-dominant brachial artery was cannulated for continuous pressure and heart-rate recording by the Oxford technique, 15 and two peripheral venous lines were inserted-one for hormone sampling, one for injection ofANP or placebo. From 0830
h to 1330 h, the subjects remained seated in an easy chair apart from brief periods of standing to pass urine every 30 min. To ensure adequate urine flow, subjects drank distilled water equal in volume to the urine passed in the previous 30 min plus 50 ml. After four baseline urine collections ANP (100 Jig) or placebo was given intravenously over 1 min in a random, double-blind experimental design. Urine collections and arterial pressure recordings were continued until 1330 h. Venous samples were obtained before andat set times after ANP or placebo administration for measurements of plasma renin activity, I6 aldosterone,17 cortisol (enzyme-linked immunosorbent assay), catecholamines, 18 antidiuretic hormone, 19 and sodium and potassium (flame photometry). As each urine sample was passed, volume, pH, and osmolality (freezing-point depression) were measured immediately, and samples were taken for determination of sodium and potassium (flame photometry), calcium and magnesium (atomic absorption spectrophotometry), phosphorus (Gomorri’s colorimetric method), and creatinine
(Beckman analyser). Blood samples from the first and second study in each volunteer were assayed for hormones in the same run. Variability within assays ranged from 4 - 9% (coefficient of variation) for aldosterone to 9-0% for adrenaline. Results from ANP and placebo studies were compared by means of the t test for paired data. Data are given as mean±standard error.
Fig 1-Urinary volume and electrolyte excretion before and after administration (arrow) of placebo (open columns) or ANP (filled columns) to six normal subjects (absolute values).
*p<0’05,
fp<0’01,
:p<0’001
for differences between ANP and
placebo.
547
2-Heart rate and mean arterial pressure integrated from continuous arterial recordings for 30 min periods before and after administration (arrow) of placebo or ANP to six normal subjects.
Fig
Results The studies were carried out without complication and data collection was complete. Mean±SEM 24 h urinary excretion of sodium and potassium was 108±5 mmol and 53±3 mmol, respectively, the day before the subjects were given placebo and 112±8 mmol and 6l:t3 mmol the day before ANP (not significant). Mean body weight did not change between the studies.
Urine There were no significant differences in urine indices between the 2 experimental days for the 30 min collection periods just before administration of ANP or placebo (fig 1). The mean urinary volume for the 30 min after ANP injection was twice that after placebo (p<0 01, fig 1) and the volumes after ANP were somewhat higher than those after placebo for the rest of the study period. Four subjects were aware of bladder distension within 10 min of receiving ANP. Urinary sodium excretion in the 30 min after infusion was four times greater after ANP than after placebo and remained significantly greater for 1’ 5 h (fig 1). Potassium excretion was
Fig 3-Systolic and diastolic arterial pressure and heart
rate for successive 1 min intervals from 10 min before to 12 min after administration (arrow) of placebo or ANP to six normal subjects.
slightly greater after ANP than after placebo, but this difference was not significant (fig 1). The urinary output of calcium, magnesium, and phosphorus was increased by ANP administration (fig 1). For calcium and magnesium the higher excretion lasted 1 h, whereas phosphorus excretion remained significantly above placebo levels for 2 h (fig 1). Urine osmolality fell steadily on both experimental days, the change being greater after ANP. However, owing to wide variations between subjects, this difference was not significant for any 30 min collection period. Urinary creatinine excretion and pH were not significantly affected by ANP. Blood Pressure and Heart Rate There was an acute rise in arterial pressure and heart rate ’ just before administration of ANP or placebo as the subjects stood to pass urine (figs 2 and 3) This was a consistent feature
MEAN PLASMA HORMONE LEVELS BEFORE AND AFTER PLACEBO
PRA= plasma
rerun
activity; ADH=antidiuretic hormone. *p<0-05
for difference between ANP and
(P) AND
placebo.
ANP
548
of the blood-pressure and heart-rate response to standing and micturition. Immediately after administration of ANP, all six subjects showed an acute fall in arterial pressure and a rise in heart rate (figs 2 and 3). In four this was associated with a short-lived sensation of flushing, and in one facial flushing was apparent to observers for approximately 1 min. The difference between ANP and placebo in arterial pressure was significant by the second minute (mean fall below placebo level 5’ 5±2’ 4 and 5 - 2± 1’ 4 mmHg in systolic and diastolic pressure, respectively), and pressures after ANP remained slightly below those after placebo for most of the next 8 min (fig 3). The difference betweenANP and placebo in heart rate also reached significance in the second minute, the maximum difference being 13’ 5± 1 4 bpm during the fifth minute after injection (fig 3). Analysis of mean data recorded over 30 min periods showed that ANP administration was associated with an initial fall in arterial pressure and a later, sustained hypotensive effect (figs 2 and 3). Similarly, heart rate increased briefly after ANP and was higher than after placebo for the rest of the study period (fig 2). Hormones There were no significant differences in plasma renin activity, aldosterone, cortisol, noradrenaline, or antidiuretic hormone between the 2 study days before or afterinjection of ANP or placebo (see table). Plasma adrenaline levels appeared to fall after ANP, though the possibility that ANP in plasma interferes with the radio-enzymic techniques of measuring adrenaline needs to be tested. Plasma sodium and potassium levels were not altered by ANP.
the renin-angiotensin-aldosterone system for maintenance of volume homeostasis. Though differences in study conditions make comparisons difficult, some of the effects ofANP resemble those ofa small intravenous bolus of frusemide. Frusemide also causes maximum increases in urine volume and sodium excretion within 30 min of administration, 2I and in primates the effects on excretion of water, sodium, and calcium are similar.6 However, acute haemodynamic effects differ. Although very low doses of frusemide increase venous capacitance within 5 min of injection, 22 systemic vascular resistance rises and arterial pressure is unchanged23,24 or may actually rise. 21,22 Levels of antidiuretic hormone,25 plasma renin activity, and aldosterone26 increase after frusemide. A direct vasodilator action, combined with acute fluid volume depletion in the absence of a vasopressor hormone response, may explain the prolonged hypotensive effect we observed after ANP. Our findings show that ANP has definite natriuretic, diuretic, and vasodepressor effects in normal man. It may also inhibit secretion from the juxtaglomerular apparatus, adrenal medulla, and zona glomerulosa. However, the type and the concentration of the circulating peptide, or peptides, in man are presently unknown. Further trials and the development of an assay for the peptide in plasma will help to show whether ANP has a role in the normal regulation of arterial pressure and volume homeostasis or in the pathophysiology and therapy of various disease states, including congestive cardiac failure and some forms of hypertension.
against
We thank ward B3 nurses, special test sisters, biochemistry and steroid laboratories, Mrs Daphne Hinton, Mr Steve Joyce, Mrs Rose Richards, and Mrs Natalie Purdue. This study was supported by the National Heart Foundation of New Zealand and the Medical Research Council of New Zealand.
Discussion These studies show that a small dose of a-ANP has clear natriuretic and vasodepressor effects in normal man. The amount administered may have been somewhat less than the 100 g (33 nmol) nominated owing to losses incurred during preparation and injection of the peptide. After injection of ANP there was a rapid, vigorous response in urine volume and electrolyte excretion. The response patterns are similar to those reported in rats.14 Whether the urinary effects can be accounted for by direct tubular actions of A-ANP, small changes in glomerular filtration rate, or redistribution of intrarenal blood flow remains to be determined. The arterial pressure response to ANP appeared to be biphasic. Mean arterial pressure fell below placebo levels within 3 min of injection. After a brief return to placebo levels at 10 min a longer vasodepressor phase followed and blood pressure remained significantly depressed (compared with the placebo day) 175 min after injection. The mechanisms underlying the hypotensive effect of ANP are not defined by our study. It seems likely, however, that the immediate effect, associated with flushing in several subjects, reflects a direct vasodilator action of ANP. The reciprocal rise in heart rate is consistent with arterial baroreceptor activation, but we did not see any substantial rise in plasma catecholamines; in fact, plasma adrenaline actually fell after ANP. Apart from the fall in plasma adrenaline, there were no clear changes in hormone levels after ANP. However, some trends consistent with data from animal experiments were seen. Aldosterone levels tended to fall in the hour after ANP, despite the concurrent diuresis and fall in blood pressure. ANP inhibits both basal and stimulated aldosterone secretion from isolated rat9 and bovine adrenal cells.2° Such an action fits well with the concept of a humoral system balanced
Correspondence should be addressed to E. A. E., Department of Endocrinology, Princess Margaret Hospital, Cashmere Road, Christchurch 2, New Zealand. REFERENCES 1. Kisch B. Electron microscopy of the atrium of the heart: guinea pig. Exp Med Surg 1956; 14: 99-112. 2. Bompiani GD, Rouiller C, Hatt PY. Le tissu de conduction du coeur chez la rat. Etude au microscope electronique. Arch Mal Coeur 1959; 52: 1257. 3. de Bold AJ. Heart atria granularity effects of changes in water-electrolyte balance. Proc Soc Exp Biol Med 1979; 161: 508-11. 4. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic
response
to intravenous
injection of atrial
myocardial extract in rats. Life Sci 1981,
28: 89-94. 5. Borenstein
atrial
HB, Cupples WA, Sonnenberg H, Veress AT. The effect of a natriutetic
extract on
Physiol 1983;
renal
haemodynamics and urinary excretion in anaesthetized rats. J
334: 133-40.
MN, Gilmore JP. Natriuretic activity of human and monkey atria. Circ Res 1983; 53: 420-23. 7. Currie MG, Geller DM, Cole BR, et al. Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria. Science 1983; 221: 71-73. 8. Kleinert HD, Maack T, Atlas SA, Januszewicz A, Sealey JE, Laragh JH. Atrial
6. Nemeh
9. 10.
11.
12.
13. 14.
15. 16.
natriuretic factor inhibits angiotensin norepinephrine, and potassium-induced vascular contractility. Hypertension 1984; 6 (suppl 1) I-143-47. Atarashi K, Mulrow P, Franco-Saenz R, Snajdar R, Rapp J. Inhibition of aldosterone production by an atrial extract. Science 1984; 224: 992-94. Chimoskey JE, Spielman WS, Brandt MA, Heidemann SR. Cardiac atria of BIO 14·6 hamsters are deficient in natriuretic factor. Science 1984; 223: 820-22. Hirata Y, Ganguli M, Tobian L, Iwai J. Dahl S rats have increased natriuretic factor in atria but are markedly hyporesponsive to it. Hypertension 1984; 6 (suppl 1). 1-148-55. Sonnenberg H, Milojevic S, Chong CK, Veress AT. Atrial natriuretic factor: reduced cardiac content in spontaneously hypertensive rats. Hypertension 1983; 5: 672-75 Oikawa S, Imai M, Ueno A, et al Cloning and sequence analysis of cDNA encoding a precursor for human atrial natriuretic polypeptide. Nature 1984; 309: 724-26. Kangawa K, Matsuo H Purification and complete ammo acid sequence of &agr;-human atrial natriuretic polypeptide (&agr;-hANP). Biochem Biophys Res Commun 1984, 118: 131-39 Millar-Craig MW, Hawes D, Whittington J. New system for recording ambulatory blood pressure in man. Med Biol Eng Comput 1978; 16: 727-31. Dunn PJ, Espiner EA. Outpatient screening tests for primary aldosteronism. Aust NZ J
Med 1976, 6: 131-35. S, Espiner EA, Nicholls MG, Yandle TG. A direct radioimmunoassay aldosterone in plasma. Clin Chem 1983; 29: 268-71.
17. Lun
for
549 had been discovered by screening in the newborn child of the first family. Increasing blood phenylalanine concentrations prompted dietary treatment from day 4 of life. The child has developed normally. One of the two children in the second family has PKU-also identified by the neonatal screening programme-and has been on a phenylalanine-restricted diet from day 9. This child is now 4 years old and has developed normally.
PRENATAL DIAGNOSIS OF CLASSIC PHENYLKETONURIA BY DNA ANALYSIS FLEMMING GÜTTLER SAVIO L. C. WOO
ALAN S. LIDSKY
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA; and John F. Kennedy Institute, Glostrup, Denmark
Howard Hughes Medical Institute,
.
Southern Blot Analysis
Prenatal diagnosis of classic
phenylketonuria (PKU) was performed in two at-risk families by means of a cloned human phenylalanine hydroxylase gene probe which was used to analyse DNA isolated from cultured amniotic fluid cells. The diagnoses of a PKU fetus in one family and a heterozygous fetus in another family were confirmed after birth. The prenatal diagnosis procedure by DNA analysis can be confidently applied to 90% of caucasian families with previously affected children. Summary
Introduction PHENYLKETONURIA
(PKU) is an inborn deficiency of the phenylalanine hydroxylase (PAH) that converts phenylalanine to tyrosine. I-3 The deficiency results in raised serum phenylalanine concentrations and mental retardation4 if the child does not receive long-term treatment with a lowphenylalanine diet5 starting within the first weeks of life.6 Mass screening of blood phenylalanine in neonates is performed in many countries. PKU is transmitted as an autosomal recessive trait,3 and it occurs in approximately 1 in 10 000 white neonates. Since the enzyme is liver-specific and is not expressed in fibroblasts, prenatal diagnosis by enzyme analysis of amniocytes is not possible. We have developed a molecular method for prenatal diagnosis of PKU by DNA analysis. We have isolated a human phenylalanine hydroxylase cDNA clone that detects the corresponding gene in the human genome.’Because several restriction endonucleases produce polymorphic patterns in the phenylalanine hydroxylase locus, the gene probe can be used to analyse PKU families by restriction fragment length polymorphism. 7,8 Since the restriction fragment patterns and the disease state were tightly linked in the families analysed, analysis of polymorphisms can theoretically lead to prenatal diagnosis of PKU in families which already have children with PKU. 7,1 We report the first two instances in which PKU was diagnosed prenatally by determining the segregation of the mutant PAH genes identified by the polymorphic restriction fragments. enzyme
Patients and Methods Patients Two families who already had one PKU child wanted to know the of their current pregnancies to prepare for delivery. PKU
outcome
18
19
Peuler JD, Johnson GA. Simultaneous single isotope radioenzymatic assays of plasma norepinephrine, epinephrine and dopamine. Life Sci 1977; 21: 625-33. Sadler WA, Lynskey CP, Gilchrist NL, Espiner EA, Nicholls MG. A sensitive radioimmunoassay for measuring plasma anti-diuretic hormone in man. NZ Med J 1983, 96: 959-63.
20 Goodfriend
TL, Elliott ME, Atlas SA Actions of synthetic atrial natriuretic factor on glomerulosa. Life Sci 1984; 35: 1675-82. 21 Andreasen F, Hansen U, Husted SE, Mogensen CE, Pedersen EB. The influence of age on renal and extrarenal effects of frusemide. Br J Clin Pharmacol 1984; 18: 65-74 22 Johnston GD, Nicholls DP, Leahey WJ. The dose-response characteristics of the acute non-diuretic peripheral vascular effects of frusemide in normal subjects. Br J Clin Pharmacol 1984; 18: 75-81 bovine adrenal
of Genomic DNA
phPAH247 is a recombinant plasmid containing a full-length human cDNA for phenylalanine hydroxylase,9 -which has been used to identify additional polymorphisms in the human PAH locus. 10 The cDNA insert was subcloned into the Eco RI site ofpBR322 and isolated by digestion of phPAH247 DNA with Eco RI followed by preparative electrophoresis in low-melting point agarose gels. The isolated DNA fragment was radiolabelled to a specific activity of 2-3 x 10g dpm/g by nick translation."Total human genomic DNA was purified from cultured amniotic-fluid cells and from peripheral-blood leucocytes isolated as buffy coats from whole blood as previously described.5g of genomic DNA was digested to completion with various restriction enzymes in appropriate buffers. Gel electrophoresis and blotting of DNA fragments to nitrocellulose filters were performed by a modified procedure of Southern. I2 The nitrocellulose filters were incubated for 5 h at 42°C in a pre-hybridisation solution containing 45% formamide, 5 x Denhardt’s solution, 4xSSC (salt and sodium citrate), 0 1 mol/1 sodium phosphate, pH 6-5, 0-1% sodium pyrophosphate, and 250 laglml sheared, denatured, herring sperm DNA. Hybridisation was performed in the pre-hybridisation solution which also contained 10% dextran sulphate and 2x 106 cpm/ml 32P-labelled PAH cDNA probe at 42°C overnight. The nitrocellulose filters were washed twice in 2 x SSC, 0’ 5% sodium dodecyl sulphate (SDS) for 15 min at room temperature, and twice in 0-lx SSC, 0-1% SDS for 2 h at 680C followed by autoradiography at -70°C for 1-5 days. Serum phenylalanine concentration was determined fluorimetrically as previously described. 13
Results Both parents in family 1 are heterozygous for the Hind III polymorphism. They both have a PAH gene containing the 4’ 2 kb fragment and a second gene containing the 4’ 0 kb fragment (fig 1, lanes 1 and 2). Digestion of DNA isolated from leucocytes of the PKU child showed that he inherited the PAH genes containing the 4’ 0 kbHind III fragment from both parents (fig 1, lane 3). Therefore, in this family the PKU traits segregated with the PAH genes containing the 4’ 0 kb fragment. Since the phenylalanine hydroxylase genes in family 1 were informative for gene analysis, DNA was isolated from cultured amniotic-fluid cells of a fetus at risk and analysed by digestion with Hind III followed by hybridisation with PAH cDNA probe. The results revealed that the fetus was homozygous for the 4 - 0 kb fragment. Thus, the fetus had the same genotype as the PKU proband; after delivery PKU was confirmed in this infant. The infant
23. Mond 24.
H, Hunt D, Sloman G. Haemodynamic effects of frusemide in patients suspected of having acute myocardial infarction. Br Heart J 1974; 36: 44-53. Lal S, Murtagh JG, Pollock AM, Fletcher E, Binnion PF. Acute haemodynamic effects of frusemide in patients with normal and raised left atrial pressures. Br Heart J 1969; 31: 711-17.
25. De Lima J, Caillens H, Beaufils M, Adaillou R. Effects of frusemide-induced plasma volume reduction on plasma antidiuretic hormone in normal and hypertensive subjects Clin Nephrol 1981; 15: 246-51.
26. Rosenthal J, Boucher R, Nowaczynski W, Genest J. Acute changes in plasma volume, renin activity, and free aldosterone levels in healthy subjects following frusemide administration Canad J Physiol Pharmacol 1968; 46: 85-91.