472
side-effect of imipramine was postural hypotension. Almost 20% of the patients were seriously affected by orthostatic hypotension. To our surprise, this effect was not, as we had expected, related to dose, nor was it limited to the elderly. It does seem likely that the potential seriousness of the effect will change with a given patient’s ability to tolerate these postural drops, and it seems reasonable to assume that, even though postural hypotension can occur at any age, the incidence of serious complications will increase with increasing age and pre-existing cardiovascular disease. Interestingly, patients at high risk for postural hypotension can be identified before they start medication by an examination of the difference between their lying and standing systolic blood-pressure before they start on the drug. We thank Dr Joseph Fleiss, Chairman, Department of Biostatistics for his thoughtful criticism, and Maxine Prince for her tireless efforts. This work was supported partly by Alcohol, Drug Abuse, and Mental Health Administration grants MH-2113 from the National Institute of Mental Health and HL-70204 from the U.S. Public Health Service. Requests for reprints should be addressed to A.H.G., Department of Psychiatry, New York State Psychiatric Institute, 722 West 168th Street, New York, N.Y. 10032.
This study was done to determine whether the levels of serum-trypsin, measured by radioimmunoassay, are abnormal in children with c.F., and to establish the assay in dried blood-spots collected for neonatal screening of other inborn errors. Patients
Blood-samples were collected from 26 children with c.F. aged 1 month to 18 years and from a variety of controls: paediatric hospital admissions with (4) and without (16) obstructive intestinal disease; 6 normal healthy children (sibs of diabetics); and 6 insulin-requiring diabetics (duration of diabetes 200 .g/1 3-5 years after birth. These were the only children studied who had some residual pancreatic function (lower pancreatin dosages and minimal steatorrhoeal; consistent with this was the presence of some pancreatic amylase in the serum (patient 6 had 60% of normal proone serum
amylase
473 One of these children (patient 7) has recently shown a clinical decline in pancreatic function, consistent with the fall in serum-i.R.T. to 180 g/1. All c.F. children older than 2 years, without residual pancreatic function, had serum-i.R.T. values similar to or below those of the other children studied. The only non-c.F. children in this study with serum-i.R.T. >200 p.g/l were those admitted to hospital with biliary or jejunal atresia.
portion).
The levels for diabetic
children (33+_24 g/1, meanis.D.) were significantly lower than for a group of sibs (84±17) and for hospital admissions (104:!:52) covering a similar age range. The mean serum-i.R.T. concentration in non-c.F., non-diabetic children without obstructive intestinal disease (104+52) was significantly below that of an adult population (272±67),3 emphasising the importance of using age-matched controls when studying serum-i.R.T. in disease states. A recent report4 of lower concentrations of serum-i.R.T. in insulin-dependent diabetics fails to distinguish between an effect of diabetes and an age effect. Our preliminary study confirms, however, that recently diagnosed diabetic children do have reduced serum-i.R.T. levels.
Dried Blood-spotAssays
*
All 11 newborn children with c.F. were clearly distinguishable from their controls and from babies with persistently low stool-trypsin (table). Because of incom-
As measured in a 150 µ1 volume of two 12 mm discs eluted in 0.5 ml buffer. t Value for c.F. or babies with low stool-trypsin, divided by means of controls.
plete elution from the discs and/or denaturation due to prolonged storage, I.R.T. values from the older cards lower in both controls and c.F. children. The results of the blood-spot assay both confirmed the serum findings and demonstrated the potential of the assay for were
establishing the diagnosis of c.F. Discussion In the first few months of life, all C.F. children studied high levels of serum-l.R.T., whether or not enzymatically active trypsin was measurable in their faeces. This supports a hypothesis that the pancreatic duct is progressively blocked with initial "back-leakage" of acinar contents into the plasma. Although spuriously
had
values of serum-l.R.T. could be obtained if comc.F. serum competed with antibody for 125I-trypsin binding, this possibility is rendered less likely by the observation that abnormally high values of serum-I.R.T. persisted only in the c.F. children with residual pancreatic function. The similarity of results obtained from dried bloodspots and from serum establishes the practicability of using a blood-I.R.T. assay as a newborn screening test for
high
ponents of
b. Children without
Cystic Fibrosis admissions
o
Hospital
e
Sibs of diabetics
A
Normal
o
Diabetics
’
(without obstructive intestinal disease)1
x
Hospital admissions (obstructive
C.F.
.
Parents with
The advantages of this test over any other previously described are (1) that the sample is already collected for other purposes in countries conducting Guthrie testing of the newborn; (2) that the test gives an abnormal result even for c.F. children with residual pancreatic function. Such c.F. children would be missed by stool-
mtestmal disease)
CF children
Serum-I.R.T. in (above) children with C.F. and (below) children without C.F.
trypsin screening.t The high cost of reagents and gamma-counting equipment is the main disadvantage of the test, but the cost of equipment would be of little consequence in centres already screening T4 or thyroid-stimulating hormone
474 for neonatal hypothyroidism. The cost of the reagents may diminish with improved technology. We thank the Medical Research Council of New Zealand for financial assistance and the department of community health, University of Auckland, for cooperation in providing access to the National File of Newborn Bloodspots.
Reprints should be addressed to J.R.C., Department of Paediatrics, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand. REFERENCES 1. 2.
Crossley, J. R., Berryman, C. C., Elliott, R. B. Lancet, 1977, ii, Frier, B. M., Saunders, J. H. B., Wormsley, K. G., Bouchier, I.
1093. A. D.
Gut,
1976, 17, 685. 3. Elias, E., Redshaw, M., Wood, T. Lancet, 1977, ii, 66. 4. Dandona, P., Elias, E., Beckett, A. G. Br. med. J. 1978, ii, 1125.
UPTAKE OF TAURINE BY PLATELETS IN RETINITIS PIGMENTOSA E. M. AIRAKSINEN* M. M. AIRAKSINEN E. TUOVINE
P. SIHVOLA M. SIHVOLA
Department of Pharmacology, University of Kuopio, and Ophthalmic and Pediatric Clinics, University Central Hospital, Kuopio, Finland
Summary Uptake of 3H-taurine by platelets from twelve patients with retinitis pigmentosa and from (R.P.) healthy controls was measured. Platelets were incubated in autologous plasma with 3H-taurine for different times and at different substrate concentrations. The uptake of taurine by R.P. platelets at each incubation time was about two-thirds of the control value, the difference being statistically significant. Km was about the same, but Vmax was lower in R.P. platelets. The results suggest that R.P. is a disease affecting not only the eye but also taurine transport and/or storage in general.
Twenty-one controls were healthy people (range 15-51, mean 31) either from our laboratory or those donating blood to the blood-bank. None of the controls or patients was taking any drugs. Methods Platelet-rich plasma (P.R.P.) was prepared according to the usual technique.9 Platelets were counted microscopically. P.R.P. was diluted with autologous platelet-free plasma to obtain 2xx 101 platelets/ml. Samples (1 ml) were prewarmed at 37°C in a water-bath, then 0.1ml of 0.11 µmol/1 3H-taurine (Radiochemical Centre, Amersham, specific activity 9.0 Ci/ mmol) was added and samples were incubated with gentle shaking for 10, 30, 60, and 120 min in an atmosphere of 95 oxygen and 5% carbon dioxide. Additional samples were incubated in an ice-bath. Duplicate samples from one, control and one to three R.P. patients were incubated simultaneously each day. For kinetic analysis, the 30 min incubation was performed with six different physiological taurine concentrations between 2.6 and 52.6 µ mol/1. Incubation was stopped by placing the test-tubes in ice. They were then centrifuged in 4 °C at 18 000 g for 10 .min. The supernatant was decanted after removal of 0.1 ml for a radioactivity count. Platelet pellets were washed with cool Krebs’ phosphate solution,9 pH 7.4, the test-tubes were dried and 0.5 ml of NCS tissue solubiliser (Amersham) and 10 ml of scintillation medium was added before radioactivity was measured.
Results Our results demonstrate that the uptake of taurine by platelets from patients with R.P.was 27-42% lower than the control values at each incubation time. A statistically significant difference was found both between the R.P. patients and their daily controls and also between R.P. patients and all healthy controls (see accompanying figure). Some samples from healthy controls were not incubated on the same day as R.P. platelets. The distribution of radioactivity between platelets and autologous plasma after incubation was significantly different in the patients and controls at each incubation-time (see accompanying table). Kinetic studies calculated according to the method of Hofstee showed that Vmax in the
Introduction
’
RETINITIS pigmentosa (R.P.) is an inheritable disease and a common cause of blindness in middle age. There is no effective treatment. Taurine is a putative neurotransmitter or neuromodulator in the retina.1-3 Lack of taurine during a critical period of development caused degenerative changes of the retinas of kittens4 which resembled those seen in R.P. These could be prevented only by administration of taurine.5 Earlier studies6,7 demonstrated that plasma-taurine concentrations are normal in R.P. patients. Platelets are often used as models of neurons in uptake studies of biogenic amines and of some aminoacids including taurine8 which is actively taken up by platelets.9 We investigated whether taurine uptake or storage are altered in patients with R.P.
Patients
I
obtained from twelve otherwise healthy outpatients with R.P., in whom diagnosis was confirmed by ophthalmologists. The mean age was 37 (range 18-60) years. Blood
was
*Present address:
138, 70101 Kuopio
Department of Pharmacology, University of Kuopio,
10, Finland.
P.O.B.
1.11
11
...
Uptake of 3H-taurine by platelets from
healthy controls. Values are given as
8 R.P.
patients
and
differences in radioactivity between samples in37° and in ice. Platelets from 5 C, controls were incubated simultaneously with R.p. samples. Those from 16 C2 controls include samples incubated at different times. Significance was determined b Student’s unpaired t test P
side-effect of imipramine was postural hypotension. Almost 20% of the patients were seriously affected by orthostatic hypotension. To our surprise, this effect was not, as we had expected, related to dose, nor was it limited to the elderly. It does seem likely that the potential seriousness of the effect will change with a given patient’s ability to tolerate these postural drops, and it seems reasonable to assume that, even though postural hypotension can occur at any age, the incidence of serious complications will increase with increasing age and pre-existing cardiovascular disease. Interestingly, patients at high risk for postural hypotension can be identified before they start medication by an examination of the difference between their lying and standing systolic blood-pressure before they start on the drug. We thank Dr Joseph Fleiss, Chairman, Department of Biostatistics for his thoughtful criticism, and Maxine Prince for her tireless efforts. This work was supported partly by Alcohol, Drug Abuse, and Mental Health Administration grants MH-2113 from the National Institute of Mental Health and HL-70204 from the U.S. Public Health Service. Requests for reprints should be addressed to A.H.G., Department of Psychiatry, New York State Psychiatric Institute, 722 West 168th Street, New York, N.Y. 10032.
This study was done to determine whether the levels of serum-trypsin, measured by radioimmunoassay, are abnormal in children with c.F., and to establish the assay in dried blood-spots collected for neonatal screening of other inborn errors. Patients
Blood-samples were collected from 26 children with c.F. aged 1 month to 18 years and from a variety of controls: paediatric hospital admissions with (4) and without (16) obstructive intestinal disease; 6 normal healthy children (sibs of diabetics); and 6 insulin-requiring diabetics (duration of diabetes 200 .g/1 3-5 years after birth. These were the only children studied who had some residual pancreatic function (lower pancreatin dosages and minimal steatorrhoeal; consistent with this was the presence of some pancreatic amylase in the serum (patient 6 had 60% of normal proone serum
amylase
473 One of these children (patient 7) has recently shown a clinical decline in pancreatic function, consistent with the fall in serum-i.R.T. to 180 g/1. All c.F. children older than 2 years, without residual pancreatic function, had serum-i.R.T. values similar to or below those of the other children studied. The only non-c.F. children in this study with serum-i.R.T. >200 p.g/l were those admitted to hospital with biliary or jejunal atresia.
portion).
The levels for diabetic
children (33+_24 g/1, meanis.D.) were significantly lower than for a group of sibs (84±17) and for hospital admissions (104:!:52) covering a similar age range. The mean serum-i.R.T. concentration in non-c.F., non-diabetic children without obstructive intestinal disease (104+52) was significantly below that of an adult population (272±67),3 emphasising the importance of using age-matched controls when studying serum-i.R.T. in disease states. A recent report4 of lower concentrations of serum-i.R.T. in insulin-dependent diabetics fails to distinguish between an effect of diabetes and an age effect. Our preliminary study confirms, however, that recently diagnosed diabetic children do have reduced serum-i.R.T. levels.
Dried Blood-spotAssays
*
All 11 newborn children with c.F. were clearly distinguishable from their controls and from babies with persistently low stool-trypsin (table). Because of incom-
As measured in a 150 µ1 volume of two 12 mm discs eluted in 0.5 ml buffer. t Value for c.F. or babies with low stool-trypsin, divided by means of controls.
plete elution from the discs and/or denaturation due to prolonged storage, I.R.T. values from the older cards lower in both controls and c.F. children. The results of the blood-spot assay both confirmed the serum findings and demonstrated the potential of the assay for were
establishing the diagnosis of c.F. Discussion In the first few months of life, all C.F. children studied high levels of serum-l.R.T., whether or not enzymatically active trypsin was measurable in their faeces. This supports a hypothesis that the pancreatic duct is progressively blocked with initial "back-leakage" of acinar contents into the plasma. Although spuriously
had
values of serum-l.R.T. could be obtained if comc.F. serum competed with antibody for 125I-trypsin binding, this possibility is rendered less likely by the observation that abnormally high values of serum-I.R.T. persisted only in the c.F. children with residual pancreatic function. The similarity of results obtained from dried bloodspots and from serum establishes the practicability of using a blood-I.R.T. assay as a newborn screening test for
high
ponents of
b. Children without
Cystic Fibrosis admissions
o
Hospital
e
Sibs of diabetics
A
Normal
o
Diabetics
’
(without obstructive intestinal disease)1
x
Hospital admissions (obstructive
C.F.
.
Parents with
The advantages of this test over any other previously described are (1) that the sample is already collected for other purposes in countries conducting Guthrie testing of the newborn; (2) that the test gives an abnormal result even for c.F. children with residual pancreatic function. Such c.F. children would be missed by stool-
mtestmal disease)
CF children
Serum-I.R.T. in (above) children with C.F. and (below) children without C.F.
trypsin screening.t The high cost of reagents and gamma-counting equipment is the main disadvantage of the test, but the cost of equipment would be of little consequence in centres already screening T4 or thyroid-stimulating hormone
474 for neonatal hypothyroidism. The cost of the reagents may diminish with improved technology. We thank the Medical Research Council of New Zealand for financial assistance and the department of community health, University of Auckland, for cooperation in providing access to the National File of Newborn Bloodspots.
Reprints should be addressed to J.R.C., Department of Paediatrics, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand. REFERENCES 1. 2.
Crossley, J. R., Berryman, C. C., Elliott, R. B. Lancet, 1977, ii, Frier, B. M., Saunders, J. H. B., Wormsley, K. G., Bouchier, I.
1093. A. D.
Gut,
1976, 17, 685. 3. Elias, E., Redshaw, M., Wood, T. Lancet, 1977, ii, 66. 4. Dandona, P., Elias, E., Beckett, A. G. Br. med. J. 1978, ii, 1125.
UPTAKE OF TAURINE BY PLATELETS IN RETINITIS PIGMENTOSA E. M. AIRAKSINEN* M. M. AIRAKSINEN E. TUOVINE
P. SIHVOLA M. SIHVOLA
Department of Pharmacology, University of Kuopio, and Ophthalmic and Pediatric Clinics, University Central Hospital, Kuopio, Finland
Summary Uptake of 3H-taurine by platelets from twelve patients with retinitis pigmentosa and from (R.P.) healthy controls was measured. Platelets were incubated in autologous plasma with 3H-taurine for different times and at different substrate concentrations. The uptake of taurine by R.P. platelets at each incubation time was about two-thirds of the control value, the difference being statistically significant. Km was about the same, but Vmax was lower in R.P. platelets. The results suggest that R.P. is a disease affecting not only the eye but also taurine transport and/or storage in general.
Twenty-one controls were healthy people (range 15-51, mean 31) either from our laboratory or those donating blood to the blood-bank. None of the controls or patients was taking any drugs. Methods Platelet-rich plasma (P.R.P.) was prepared according to the usual technique.9 Platelets were counted microscopically. P.R.P. was diluted with autologous platelet-free plasma to obtain 2xx 101 platelets/ml. Samples (1 ml) were prewarmed at 37°C in a water-bath, then 0.1ml of 0.11 µmol/1 3H-taurine (Radiochemical Centre, Amersham, specific activity 9.0 Ci/ mmol) was added and samples were incubated with gentle shaking for 10, 30, 60, and 120 min in an atmosphere of 95 oxygen and 5% carbon dioxide. Additional samples were incubated in an ice-bath. Duplicate samples from one, control and one to three R.P. patients were incubated simultaneously each day. For kinetic analysis, the 30 min incubation was performed with six different physiological taurine concentrations between 2.6 and 52.6 µ mol/1. Incubation was stopped by placing the test-tubes in ice. They were then centrifuged in 4 °C at 18 000 g for 10 .min. The supernatant was decanted after removal of 0.1 ml for a radioactivity count. Platelet pellets were washed with cool Krebs’ phosphate solution,9 pH 7.4, the test-tubes were dried and 0.5 ml of NCS tissue solubiliser (Amersham) and 10 ml of scintillation medium was added before radioactivity was measured.
Results Our results demonstrate that the uptake of taurine by platelets from patients with R.P.was 27-42% lower than the control values at each incubation time. A statistically significant difference was found both between the R.P. patients and their daily controls and also between R.P. patients and all healthy controls (see accompanying figure). Some samples from healthy controls were not incubated on the same day as R.P. platelets. The distribution of radioactivity between platelets and autologous plasma after incubation was significantly different in the patients and controls at each incubation-time (see accompanying table). Kinetic studies calculated according to the method of Hofstee showed that Vmax in the
Introduction
’
RETINITIS pigmentosa (R.P.) is an inheritable disease and a common cause of blindness in middle age. There is no effective treatment. Taurine is a putative neurotransmitter or neuromodulator in the retina.1-3 Lack of taurine during a critical period of development caused degenerative changes of the retinas of kittens4 which resembled those seen in R.P. These could be prevented only by administration of taurine.5 Earlier studies6,7 demonstrated that plasma-taurine concentrations are normal in R.P. patients. Platelets are often used as models of neurons in uptake studies of biogenic amines and of some aminoacids including taurine8 which is actively taken up by platelets.9 We investigated whether taurine uptake or storage are altered in patients with R.P.
Patients
I
obtained from twelve otherwise healthy outpatients with R.P., in whom diagnosis was confirmed by ophthalmologists. The mean age was 37 (range 18-60) years. Blood
was
*Present address:
138, 70101 Kuopio
Department of Pharmacology, University of Kuopio,
10, Finland.
P.O.B.
1.11
11
...
Uptake of 3H-taurine by platelets from
healthy controls. Values are given as
8 R.P.
patients
and
differences in radioactivity between samples in37° and in ice. Platelets from 5 C, controls were incubated simultaneously with R.p. samples. Those from 16 C2 controls include samples incubated at different times. Significance was determined b Student’s unpaired t test P
