- Email: [email protected]
Serum sulfate levels in patients with cystic fibrosis
ClinicaChimica Acta, 142 (1984) 241-247
241
Elsevier CCA 02963
Serum sulfate levels in patients with cystic fibrosis D e n n i s J. P i l l i o n *, T o n i T. N e u m e i e r a n d E l i a s M e e z a n Department of PharmacoloKv, Universit_v of Alabama in Birmingham, Birmingham, A L 35294 ( USA )
(Received March 14th, 1984; revision June 1st, 1984) Key words." Cvstic fibrosis; Sulfate levels; Ion chromatography
Summary Patients with cystic fibrosis ( C F ) secrete copious a m o u n t s of m u c o u s material which is viscous, tends to a c c u m u l a t e in the r e s p i r a t o r y tract a n d contains larger than n o r m a l a m o u n t s of sulfate. The present investigation was designed to measure sulfate levels in the serum of patients with cystic fibrosis by ion c h r o m a t o g r a p h y of protein-free serum aliquots. The level of inorganic sulfate in the serum of non-cystic fibrosis pediatric patients averaged 0.29 _+ 0.03 m m o l / l while patients suffering from cystic fibrosis had an average serum sulfate value of 0.27 _+ 0.03 m m o l / 1 which was not significantly different from controls. N o differences were observed in serum sulfate levels a m o n g males and females of either g r o u p of patients. There was a t e n d e n c y for serum sulfate levels to decrease with age, b u t there was no statistically significant difference in serum sulfate levels between cystic fibrosis patients and n o r m a l s as a function of age. These findings indicate that the highly sulfated m u c o i d materials secreted b y cystic fibrosis patients are not reflected in a b n o r m a l serum sulfate levels.
Introduction Cystic fibrosis ( C F ) is an inherited d i s o r d e r characterized by the a c c u m u l a t i o n of viscous m u c o i d material in the lungs a n d recurrent episodes of r e s p i r a t o r y infections. I m p r o v e d t r e a t m e n t m o d a l i t i e s have e x t e n d e d the life-expectancy of C F patients, but little is k n o w n a b o u t the underlying cause of the disease a n d research into this p r o b l e m has been h a m p e r e d b y the lack of an a p p r o p r i a t e l a b o r a t o r y a n i m a l m o d e l system which a p p r o x i m a t e s the h u m a n condition. Some progress has been m a d e
* To whom correspondence and reprint requests should be addressed. 0009-8981/84/$03.00 0~, 1984 Elsevier Science Publishers B.V.
24:
recently
in analysis
sputum.
It
has
glycoproteins
of the
been
by
the
longer
in which
compared
to normal
biochemical
shown mucous
Boat
composition
of the
and
[l]
that
chains
are
Cheng
carbohydrate
components.
Frates
the process
nor is much Suzuki chick
and
sternal
in tissue
tissue
sulfate
secretory
products. to
was utilized
levels
were
and
as first
Sera from
Striver
were
obtained
were obtained
from
from
(6 female.
female,
volunteers
19 male. (6 females,
because
in
biological
it is
bariulm.
to this problem
chromatography
using
01 ()I
fluids.
radiolabelled
measured
followed
hy
the level
of
this technique.
patients
of serum
acetonitrile and
12 to
used
for sulfate phosphate
serum.
were
Blanks
18 yr);
17 in-patients
tube
and
was
distilled
then
cases,
used
in place
water
alone.
The D-10 Ion Analyzer (Dionex, Sunnyvale, anion exchange column (3 x 500 mm) consisting tetramethyl
at the local
ammonium
groups
attached.
in age
children’s clinic
nursing
school
student
restrictions
were
imposcc
an equal
centrifuged
diluted
In some
with
ranging
at - 20 ‘C. Non-C‘I‘
at the adolescent
10 healthy
subsequently
was
analyses. buffer
run with
and
0.5 ml) was mixed
supernatant
Krebs-Ringer
groups:
11 males)
and stored
age 18 to 30 yr). No dietary
(usually
in a microfuge
The protein-free
(21 females,
age 1 to 16 yr); 32 out-patients
4 male,
An aliquot
with
routinely
[6]. We have
by venipuncture
the following
age
collection.
water
anion
in sthe ie\,eta
sulfation
A new approach
and in controls
32 CF
11 male,
sample
min.
high
increased
performed with
protecj-
and methods
hospital
before
to
of both
of sulfate
unusually
by precipitation used
step\.
cartilage
in the level
found
by Anderson
glycoproteins.
of sulfation
anions
[5], who
described
sulfat.ed
respirator\
at these
epiphyseal
lead
fast nor economical.
1 to 25 yr. Sera were collected
sera
would
other
measured
was neither
and
or not
are not
the
highly that
tract
process
by a decrease
levels
from
in the sera of CF patients
Materials
(13
sulfate
the
whether
in CF secrete
that were more acidic and tissues. Very little is known
intervention
that
medium
found patients
very
of respiratory
sulfate
known
culture
sulfate
by Cole
conductimetry, sulfate
the
of serum
sulfate
shown
chondroitin
It is not
separate
but this technique
sulfation
pharmacological
can be reduced
in
Measurements Previously,
cartilage
medium.
inorganic
and
possible
[3,4] have
culture
culture
difficult
about
co-workers
embryo
glycan
of glycosylation
known
CF
et al [2] found
epithelial tissues from CF patients secreted glycoproteins more highly sulfated than those secreted from non-CF about
material
volume
of 1004
at 12 500 >: ,r: for 7
with four volumes of distilled a solution of sodium sulfate or of the
serum
or added
CA. USA) was equipped of styrene/divinylbenzene Anions
are eluted
from
to the with an beads
the column
with a buffer containing 3 mmol/l NaHCO, and 2.4 mmol/l Na,CO, at a flow rate of 138 ml/h. Under these conditions, sulfate elutes from the column after 17 min with a minimal detectable limit of 0.005 mmol/l. A standard curve was constructed using
a series
the column
of sodium lost efficiency
sulfate with
solutions
of increasing
time
had
and
strength.
to be replaced
It was observed after
150-200
that
samples
243
were analyzed. Comparable results were obtained when serum proteins were removed by ultrafiltration.
Results The analysis of a solution of 0.25 m m o l / l sodium sulfate by ion chromatography is depicted in Fig. IA. It can be seen that only one significant peak is observed and that this peak appears approximately 17 rain after the sample is injected. When a 0.50 mmol/1 sodium sulfate solution is analyzed (Fig. 1B), the height of the peak observed increases from 8 to 16 chart units, confirming a linear response between ion concentration and peak height. Routinely a standard curve was constructed using four sodium sulfate solutions of increasing strength. When other ions are added to a sodium sulfate solution, there is no interference (Fig. 2). It can also be seen that sulfate is retained on the ion exchange column longer than any of the other anions tested, making quantitation of the peak height of this anion unambiguous. Further evidence that the ion chromatograph is able to separate and quantitate sulfate ions in a complex solution is presented in Fig. 3. Human serum was collected
A
n~i-
B
o
a.. i
i
LL
I'~, ~11 i o
0
~0
20 Minutes
0
]b
20
0
~0
20
Minutes
Fig. 1. Analysis of sulfate levels by anion chromatography. Solutions containing 0.25 mmol/1 sodium sulfate (A) or 0.50 mmol/1 sodium sulfate (B) were diluted with water and applied to the Dionex Ion Chromatograph equipped with an anion exchange column. Variability in peak height or in the time of peak appearance was consistently < 5%. Fig. 2. Discrimination of anions by ion chromatography. A standard solution containing a mixture of anions was diluted with water and applied to the ion chromatograph. Samples treated with acetonitrile gave an identical result.
244 b y v e n i p u n c t u r e a n d m i x e d w i t h 100% a c e t o n i t r i l e in a 1 : 1 r a t i o to p r e c i p i t a t e proteins. An aliquot of the supernatant was diluted with 3 volumes of water and a n a l y z e d o n t h e i o n c h r o m a t o g r a p h , as s h o w n in Fig. 3A. It c a n b e s e e n t h a t t h e r e a r e h i g h levels o f f l u o r i d e a n d e s p e c i a l l y c h l o r i d e in t h e s e r u m , a l o n g w i t h a moderate amount of phosphate and sulfate and only trace amounts of nitrite, nitrate o r b r o m i d e . W h e n a s o l u t i o n of s o d i u m s u l f a t e w a s a d d e d to a n i d e n t i c a l a l i q u o t of t h i s p r o t e i n - f r e e s e r u m s u p e r n a t a n t a n d a n a l y z e d b y i o n c h r o m a t o g r a p h y (Fig. 3B), t h e r e w a s a n i n c r e a s e in t h e p e a k h e i g h t c o r r e s p o n d i n g to t h e p o s i t i o n of t h e s u l f a t e ion and no other changes were observed. The height of the sulfate peak increased f r o m 4.7 to 8.05 c h a r t u n i t s a n d t h i s c o r r e s p o n d s to a n a c t u a l i n c r e a s e o f 103% o f 1he t h e o r e t i c a l v a l u e e x p e c t e d for t h i s s o l u t i o n . In a n o t h e r e x p e r i m e n t , s o d i u m s u l f a t e w a s a d d e d to a n a l i q u o t o f h u m a n s e r u m b e f o r e p r o t e i n r e m o v a l w i t h a c e t o n i t r i l e . A n a l y s i s o f t h e s u l f a t e p e a k h e i g h t s in t h e s a m p l e s w i t h a n d w i t h o u t e x o g e n o u s l ~ a d d e d s o d i u m s u l f a t e r e v e a l e d a 99% r e c o v e r y o f t h e s u l f a t e a d d e d to t h e s e r u m . These results confirm the direct relationship between peak height and sulfate concentration and suggest that human serum does not contain any components that
F ! Non
~'F
CF
0 45" 040 o
~035
20 Minute
~
°~o
E
,~ 025~ ©.20 m
.'.~
olgoo %gg
"~ 0 . 1 5 -
]0
20
O IO-
Fig. 3. Analysis of human serum sulfate levels. Human serum was added to 100% acetonitrile in a 1 I ratio and precipitated proteins were removed by centrifugation. The supernatant solution was diluted with water (A) or diluted with a standard solution of sodium sulfate (BI and applied to the ion chromatograph. Fig. 4. Serum sulfate levels in CF and non-CF pediatric patients. Serum from 17 non-CF patients. {10 males, 7 females) and 31 CF patients (10 males, 21 females) was mixed with acetonitrile to precipitate proteins and then diluted with water and analyzed on the ion chromatograph. Sulfate levels were calculated from peak heights compared to a standard curve constructed with solutions of sodium sulfate. Bars represent the means + SEM for both sexes. Males e, Females O.
245 interfere with the analysis of inorganic sulfate by ion c h r o m a t o g r a p h y . The level of inorganic sulfate in h u m a n sera collected from patients at a local children's hospital is d e p i c t e d in Fig. 4. It can be seen that n o n - C F patients have a mean serum sulfate level of 0.29 m m o l / 1 with a range of 0.21 to 0.42 m m o l / l . Five patients suffering from renal failure showed elevated serum sulfate levels (0.47 to 1.39 m m o l / 1 ) and their values have been excluded from this study. Patients suffering from C F had a mean serum sulfate level of 0.27 retool, with a range of 0.13 to 0.46 m m o l / l . One difference between these two p o p u l a t i o n s of patients was that several of the C F patients were older than any of the n o n - C F patients tested. Since there is evidence from the study of Cole and Scriver [7] that serum sulfate levels are elevated at birth and decline through the first three years of life, we o b t a i n e d a d d i t i o n a l a g e - m a t c h e d n o n - C F sera and analyzed the d a t a with respect to the age of the individual. It can be seen in Fig. 5 that the serum sulfate level in n o n - C F patients does tend to decrease with age although there is no sharp d e l i n e a t i o n between individuals above a n d below the age of three, as described by Cole and Scriver [7]. Analysis of the d a t a o b t a i n e d with C F patients is difficult to resolve unequivocally and there is insufficient evidence to interpret the level of serum sulfate as a function of age between 0 and 3 yr of age. There is c o n s i d e r a b l e overlap of the serum sulfate level measured in n o n - C F and C F patients at every age a n d there is no statistically significant difference between the values o b t a i n e d with males versus females of either the n o n - C F or the C F groups. A n o t h e r classification of pediatric p o p u l a t i o n s which could be significant in this work is that of the Sexual M a t u r i t y Index. It is known that C F patients have delayed onset of p u b e r t y a n d this could be associated with a change in the level of serum sulfate in a g e - m a t c h e d individuals. W h e n b o t h n o n - C F a n d C F patients are divided according to their Sexual M a t u r i t y Index, however, there is no significant difference between the level of sulfate in the sera of n o n - C F and C F patients at any stage.
Non-CF
o.41 o31
•
°
E o~] CF
j 03
o21
e
~o °° o:
•
.o. o'.
O1. lb
15 20 AGE (yr)
25
30
Fig. 5. Serum sulfate levels in patients of various ages. Sera from 59 non-CF patients (34 males, 25 females) and 27 CF patients (11 males, 16 females) were analyzed on the ion chromatograph for sulfate content as described in the text and in Fig. 4. Males e, Females O.
246
Discussion Cystic fibrosis occurs with high frequency among
humans,
but no genetically
c o m p a r a b l e a n i m a l m o d e l for t h e d i s e a s e is a v a i l a b l e . A t p r e s e n t , we c a n o n l y g a t h e r as m u c h i n f o r m a t i o n as p o s s i b l e o n t h e b i o c h e m i c a l p r o p e r t i e s o f C F tissues a n d h o p e t h a t t h e r e will b e a n i m p r o v e m e n t in o u r u n d e r s t a n d i n g of t h e d i s e a s e procc~,s o r a p h a r m a c o l o g i c a l b r e a k t h r o u g h in the t r e a t m e n t o f C F . C F s w e a t c o n t a i n s h i g h levels of c h l o r i d e b u t n o t h i n g is k n o w n a b o u t the level o f s u l f a t e in this s e c r e t i o n , d e s p i t e t h e fact t h a t c h l o r i d e a n d s u l f a t e are t h o u g h t to b e t r a n s p o r t e d a c r o s s cell m e m b r a n e s via t h e s a m e c a r r i e r . It is c o n c e i v a b l e t h a t the b a s i c d e f e c t in the s e c r e t o r y tissues o f C F p a t i e n t s is a n a l t e r a t i o n in t h e a c t i v i t y o f t h e a n i o n c a r r i e r p r o t e i n , l e a d i n g to e x c e s s i v e s e c r e t i o n o f c h l o r i d e a n d a c o n c o m i t a n t d e c r e a s e in s u l f a t e e x c r e t i o n o r a c h a n g e in i n t r a c e l l u l a r s u l f a t e levels. S u c h a s i t u a t i o n c o u l d l e a d to e i t h e r a d e p l e t i o n o f s e r u m s u l f a t e levels d u e to t i s s u e s e q u e s t r a t i o n of t h e ion o r a n e l e v a t i o n o f s e r u m s u l f a t e levels a s s o c i a t e d w i t h i m p a i r e d u p t a k e of s u l f a t e b y C F tissues. A l t e r n a t i v e l y , c o m p e n s a t o r y c h a n g e s in a b s o r p t i o n a n d e x c r e t i o n c o u l d p r o d u c e a s t a t e of n o r m o s u l f a t e m i a e v e n in t h e face of s e q u e s t r a t i o n of t h e a n i o n in s e c r e t o r y tissues a n d t h e e v e n t u a l loss of s u l f a t e via t h e release of h i g h l y - s u l f a t e d s e c r e t o r y p r o d u c t s . T h e r e s u l t s of t h e c u r r e n t s t u d y e x t e n d o u r k n o w l e d g e a b o u t the level of s u l f a t e in t h e s e r u m o f C F a n d n o n - C F p a t i e n t s , w i t h n o sexual or CF-related differences documented.
Acknowledgements T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m t h e C y s t i c F i b r o s i s F o u n d a t i o n . T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e t h e a s s i s t a n c e of Dr. R i c h a r d T h o m p s o n , D e p t . o f P u b l i c H e a l t h , U . A . B . in t h e p e r f o r m a n c e o f ion c h r o m a t o g r a p h y . Drs. R a y m o n d L y r e n e a n d R a l p h T i l l e r a n d Ms. M a r y B a u c o m o f the U . A . B . C y s t i c F i b r o s i s C e n t e r a n d Dr. W i l l i a m D a n i e l a n d Ms. A n n e T u r n e r a r e t h a n k e d for the c o l l e c t i o n o f sera f r o m C F a n d n o n - C F p a t i e n t s .
References I Boat TF, Cheng P. Cystic fibrosis. In: Mangos JA, Talamo RC, eds. New York: Stratton Intercontinental Medical Book Corp., 1976: 165-178. 2 Frates RC, Kaizu TT, Last JA. Mucus glycoproteins secreted by respirator3' epithelial tissue from cystic fibrosis patients. Pediat Res 1983; 17:30 34, 3 ho K. Kimata K, Sobue M, Suzuki S. Altered proteoglycan synthesis by epiphyseal cartilages m culture at low SO4- concentration. J Biol Chem 1982; 257:917 923 4 Sobue M, Takeuchi J, Ito K, Kimata K, Suzuki S. Effect of environmental sulfate concentrahons on the synthesis of low and high sulfated chondroitin sulfates by chick embryo cartilage I Biol Chem 1978: 253: 6190-6196. 5 Cole DEC, Striver CR. Microassay of inorganic sulfate in biological fluids by controlled flo,x amon chromatography. J Chromatogr 1981; 225: 359-367. 6 Anderson C. Ion chromatography: a new technique for clinical chemistry. Clin ('hem lt~7~< 22: 1424-1426. 7 Cole DEC, Scriver CR. Age-dependent serum sulfate levels in children and adolescents. Clin ('him Acta 1980; 107: 135-139.
247 8 Jennings ML, Passow 1|. Anion transport across the erythrocyte membrane, in situ proleolysis at band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate. Biochim Biophys Acta 1979; 554:498 519. 9 I,evmson C. Chloride and sulfate transport in Ehrlich ascites tumor cells: evidence for a common transport mechanism. J Cell Physiol 1978: 95:23 32. 10 Ramjeesingh M, Gaarn A, Rothslein A. The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red cell membrane. Biochim Biophys Acta 1980; 599:127 139.
241
Elsevier CCA 02963
Serum sulfate levels in patients with cystic fibrosis D e n n i s J. P i l l i o n *, T o n i T. N e u m e i e r a n d E l i a s M e e z a n Department of PharmacoloKv, Universit_v of Alabama in Birmingham, Birmingham, A L 35294 ( USA )
(Received March 14th, 1984; revision June 1st, 1984) Key words." Cvstic fibrosis; Sulfate levels; Ion chromatography
Summary Patients with cystic fibrosis ( C F ) secrete copious a m o u n t s of m u c o u s material which is viscous, tends to a c c u m u l a t e in the r e s p i r a t o r y tract a n d contains larger than n o r m a l a m o u n t s of sulfate. The present investigation was designed to measure sulfate levels in the serum of patients with cystic fibrosis by ion c h r o m a t o g r a p h y of protein-free serum aliquots. The level of inorganic sulfate in the serum of non-cystic fibrosis pediatric patients averaged 0.29 _+ 0.03 m m o l / l while patients suffering from cystic fibrosis had an average serum sulfate value of 0.27 _+ 0.03 m m o l / 1 which was not significantly different from controls. N o differences were observed in serum sulfate levels a m o n g males and females of either g r o u p of patients. There was a t e n d e n c y for serum sulfate levels to decrease with age, b u t there was no statistically significant difference in serum sulfate levels between cystic fibrosis patients and n o r m a l s as a function of age. These findings indicate that the highly sulfated m u c o i d materials secreted b y cystic fibrosis patients are not reflected in a b n o r m a l serum sulfate levels.
Introduction Cystic fibrosis ( C F ) is an inherited d i s o r d e r characterized by the a c c u m u l a t i o n of viscous m u c o i d material in the lungs a n d recurrent episodes of r e s p i r a t o r y infections. I m p r o v e d t r e a t m e n t m o d a l i t i e s have e x t e n d e d the life-expectancy of C F patients, but little is k n o w n a b o u t the underlying cause of the disease a n d research into this p r o b l e m has been h a m p e r e d b y the lack of an a p p r o p r i a t e l a b o r a t o r y a n i m a l m o d e l system which a p p r o x i m a t e s the h u m a n condition. Some progress has been m a d e
* To whom correspondence and reprint requests should be addressed. 0009-8981/84/$03.00 0~, 1984 Elsevier Science Publishers B.V.
24:
recently
in analysis
sputum.
It
has
glycoproteins
of the
been
by
the
longer
in which
compared
to normal
biochemical
shown mucous
Boat
composition
of the
and
[l]
that
chains
are
Cheng
carbohydrate
components.
Frates
the process
nor is much Suzuki chick
and
sternal
in tissue
tissue
sulfate
secretory
products. to
was utilized
levels
were
and
as first
Sera from
Striver
were
obtained
were obtained
from
from
(6 female.
female,
volunteers
19 male. (6 females,
because
in
biological
it is
bariulm.
to this problem
chromatography
using
01 ()I
fluids.
radiolabelled
measured
followed
hy
the level
of
this technique.
patients
of serum
acetonitrile and
12 to
used
for sulfate phosphate
serum.
were
Blanks
18 yr);
17 in-patients
tube
and
was
distilled
then
cases,
used
in place
water
alone.
The D-10 Ion Analyzer (Dionex, Sunnyvale, anion exchange column (3 x 500 mm) consisting tetramethyl
at the local
ammonium
groups
attached.
in age
children’s clinic
nursing
school
student
restrictions
were
imposcc
an equal
centrifuged
diluted
In some
with
ranging
at - 20 ‘C. Non-C‘I‘
at the adolescent
10 healthy
subsequently
was
analyses. buffer
run with
and
0.5 ml) was mixed
supernatant
Krebs-Ringer
groups:
11 males)
and stored
age 18 to 30 yr). No dietary
(usually
in a microfuge
The protein-free
(21 females,
age 1 to 16 yr); 32 out-patients
4 male,
An aliquot
with
routinely
[6]. We have
by venipuncture
the following
age
collection.
water
anion
in sthe ie\,eta
sulfation
A new approach
and in controls
32 CF
11 male,
sample
min.
high
increased
performed with
protecj-
and methods
hospital
before
to
of both
of sulfate
unusually
by precipitation used
step\.
cartilage
in the level
found
by Anderson
glycoproteins.
of sulfation
anions
[5], who
described
sulfat.ed
respirator\
at these
epiphyseal
lead
fast nor economical.
1 to 25 yr. Sera were collected
sera
would
other
measured
was neither
and
or not
are not
the
highly that
tract
process
by a decrease
levels
from
in the sera of CF patients
Materials
(13
sulfate
the
whether
in CF secrete
that were more acidic and tissues. Very little is known
intervention
that
medium
found patients
very
of respiratory
sulfate
known
culture
sulfate
by Cole
conductimetry, sulfate
the
of serum
sulfate
shown
chondroitin
It is not
separate
but this technique
sulfation
pharmacological
can be reduced
in
Measurements Previously,
cartilage
medium.
inorganic
and
possible
[3,4] have
culture
culture
difficult
about
co-workers
embryo
glycan
of glycosylation
known
CF
et al [2] found
epithelial tissues from CF patients secreted glycoproteins more highly sulfated than those secreted from non-CF about
material
volume
of 1004
at 12 500 >: ,r: for 7
with four volumes of distilled a solution of sodium sulfate or of the
serum
or added
CA. USA) was equipped of styrene/divinylbenzene Anions
are eluted
from
to the with an beads
the column
with a buffer containing 3 mmol/l NaHCO, and 2.4 mmol/l Na,CO, at a flow rate of 138 ml/h. Under these conditions, sulfate elutes from the column after 17 min with a minimal detectable limit of 0.005 mmol/l. A standard curve was constructed using
a series
the column
of sodium lost efficiency
sulfate with
solutions
of increasing
time
had
and
strength.
to be replaced
It was observed after
150-200
that
samples
243
were analyzed. Comparable results were obtained when serum proteins were removed by ultrafiltration.
Results The analysis of a solution of 0.25 m m o l / l sodium sulfate by ion chromatography is depicted in Fig. IA. It can be seen that only one significant peak is observed and that this peak appears approximately 17 rain after the sample is injected. When a 0.50 mmol/1 sodium sulfate solution is analyzed (Fig. 1B), the height of the peak observed increases from 8 to 16 chart units, confirming a linear response between ion concentration and peak height. Routinely a standard curve was constructed using four sodium sulfate solutions of increasing strength. When other ions are added to a sodium sulfate solution, there is no interference (Fig. 2). It can also be seen that sulfate is retained on the ion exchange column longer than any of the other anions tested, making quantitation of the peak height of this anion unambiguous. Further evidence that the ion chromatograph is able to separate and quantitate sulfate ions in a complex solution is presented in Fig. 3. Human serum was collected
A
n~i-
B
o
a.. i
i
LL
I'~, ~11 i o
0
~0
20 Minutes
0
]b
20
0
~0
20
Minutes
Fig. 1. Analysis of sulfate levels by anion chromatography. Solutions containing 0.25 mmol/1 sodium sulfate (A) or 0.50 mmol/1 sodium sulfate (B) were diluted with water and applied to the Dionex Ion Chromatograph equipped with an anion exchange column. Variability in peak height or in the time of peak appearance was consistently < 5%. Fig. 2. Discrimination of anions by ion chromatography. A standard solution containing a mixture of anions was diluted with water and applied to the ion chromatograph. Samples treated with acetonitrile gave an identical result.
244 b y v e n i p u n c t u r e a n d m i x e d w i t h 100% a c e t o n i t r i l e in a 1 : 1 r a t i o to p r e c i p i t a t e proteins. An aliquot of the supernatant was diluted with 3 volumes of water and a n a l y z e d o n t h e i o n c h r o m a t o g r a p h , as s h o w n in Fig. 3A. It c a n b e s e e n t h a t t h e r e a r e h i g h levels o f f l u o r i d e a n d e s p e c i a l l y c h l o r i d e in t h e s e r u m , a l o n g w i t h a moderate amount of phosphate and sulfate and only trace amounts of nitrite, nitrate o r b r o m i d e . W h e n a s o l u t i o n of s o d i u m s u l f a t e w a s a d d e d to a n i d e n t i c a l a l i q u o t of t h i s p r o t e i n - f r e e s e r u m s u p e r n a t a n t a n d a n a l y z e d b y i o n c h r o m a t o g r a p h y (Fig. 3B), t h e r e w a s a n i n c r e a s e in t h e p e a k h e i g h t c o r r e s p o n d i n g to t h e p o s i t i o n of t h e s u l f a t e ion and no other changes were observed. The height of the sulfate peak increased f r o m 4.7 to 8.05 c h a r t u n i t s a n d t h i s c o r r e s p o n d s to a n a c t u a l i n c r e a s e o f 103% o f 1he t h e o r e t i c a l v a l u e e x p e c t e d for t h i s s o l u t i o n . In a n o t h e r e x p e r i m e n t , s o d i u m s u l f a t e w a s a d d e d to a n a l i q u o t o f h u m a n s e r u m b e f o r e p r o t e i n r e m o v a l w i t h a c e t o n i t r i l e . A n a l y s i s o f t h e s u l f a t e p e a k h e i g h t s in t h e s a m p l e s w i t h a n d w i t h o u t e x o g e n o u s l ~ a d d e d s o d i u m s u l f a t e r e v e a l e d a 99% r e c o v e r y o f t h e s u l f a t e a d d e d to t h e s e r u m . These results confirm the direct relationship between peak height and sulfate concentration and suggest that human serum does not contain any components that
F ! Non
~'F
CF
0 45" 040 o
~035
20 Minute
~
°~o
E
,~ 025~ ©.20 m
.'.~
olgoo %gg
"~ 0 . 1 5 -
]0
20
O IO-
Fig. 3. Analysis of human serum sulfate levels. Human serum was added to 100% acetonitrile in a 1 I ratio and precipitated proteins were removed by centrifugation. The supernatant solution was diluted with water (A) or diluted with a standard solution of sodium sulfate (BI and applied to the ion chromatograph. Fig. 4. Serum sulfate levels in CF and non-CF pediatric patients. Serum from 17 non-CF patients. {10 males, 7 females) and 31 CF patients (10 males, 21 females) was mixed with acetonitrile to precipitate proteins and then diluted with water and analyzed on the ion chromatograph. Sulfate levels were calculated from peak heights compared to a standard curve constructed with solutions of sodium sulfate. Bars represent the means + SEM for both sexes. Males e, Females O.
245 interfere with the analysis of inorganic sulfate by ion c h r o m a t o g r a p h y . The level of inorganic sulfate in h u m a n sera collected from patients at a local children's hospital is d e p i c t e d in Fig. 4. It can be seen that n o n - C F patients have a mean serum sulfate level of 0.29 m m o l / 1 with a range of 0.21 to 0.42 m m o l / l . Five patients suffering from renal failure showed elevated serum sulfate levels (0.47 to 1.39 m m o l / 1 ) and their values have been excluded from this study. Patients suffering from C F had a mean serum sulfate level of 0.27 retool, with a range of 0.13 to 0.46 m m o l / l . One difference between these two p o p u l a t i o n s of patients was that several of the C F patients were older than any of the n o n - C F patients tested. Since there is evidence from the study of Cole and Scriver [7] that serum sulfate levels are elevated at birth and decline through the first three years of life, we o b t a i n e d a d d i t i o n a l a g e - m a t c h e d n o n - C F sera and analyzed the d a t a with respect to the age of the individual. It can be seen in Fig. 5 that the serum sulfate level in n o n - C F patients does tend to decrease with age although there is no sharp d e l i n e a t i o n between individuals above a n d below the age of three, as described by Cole and Scriver [7]. Analysis of the d a t a o b t a i n e d with C F patients is difficult to resolve unequivocally and there is insufficient evidence to interpret the level of serum sulfate as a function of age between 0 and 3 yr of age. There is c o n s i d e r a b l e overlap of the serum sulfate level measured in n o n - C F and C F patients at every age a n d there is no statistically significant difference between the values o b t a i n e d with males versus females of either the n o n - C F or the C F groups. A n o t h e r classification of pediatric p o p u l a t i o n s which could be significant in this work is that of the Sexual M a t u r i t y Index. It is known that C F patients have delayed onset of p u b e r t y a n d this could be associated with a change in the level of serum sulfate in a g e - m a t c h e d individuals. W h e n b o t h n o n - C F a n d C F patients are divided according to their Sexual M a t u r i t y Index, however, there is no significant difference between the level of sulfate in the sera of n o n - C F and C F patients at any stage.
Non-CF
o.41 o31
•
°
E o~] CF
j 03
o21
e
~o °° o:
•
.o. o'.
O1. lb
15 20 AGE (yr)
25
30
Fig. 5. Serum sulfate levels in patients of various ages. Sera from 59 non-CF patients (34 males, 25 females) and 27 CF patients (11 males, 16 females) were analyzed on the ion chromatograph for sulfate content as described in the text and in Fig. 4. Males e, Females O.
246
Discussion Cystic fibrosis occurs with high frequency among
humans,
but no genetically
c o m p a r a b l e a n i m a l m o d e l for t h e d i s e a s e is a v a i l a b l e . A t p r e s e n t , we c a n o n l y g a t h e r as m u c h i n f o r m a t i o n as p o s s i b l e o n t h e b i o c h e m i c a l p r o p e r t i e s o f C F tissues a n d h o p e t h a t t h e r e will b e a n i m p r o v e m e n t in o u r u n d e r s t a n d i n g of t h e d i s e a s e procc~,s o r a p h a r m a c o l o g i c a l b r e a k t h r o u g h in the t r e a t m e n t o f C F . C F s w e a t c o n t a i n s h i g h levels of c h l o r i d e b u t n o t h i n g is k n o w n a b o u t the level o f s u l f a t e in this s e c r e t i o n , d e s p i t e t h e fact t h a t c h l o r i d e a n d s u l f a t e are t h o u g h t to b e t r a n s p o r t e d a c r o s s cell m e m b r a n e s via t h e s a m e c a r r i e r . It is c o n c e i v a b l e t h a t the b a s i c d e f e c t in the s e c r e t o r y tissues o f C F p a t i e n t s is a n a l t e r a t i o n in t h e a c t i v i t y o f t h e a n i o n c a r r i e r p r o t e i n , l e a d i n g to e x c e s s i v e s e c r e t i o n o f c h l o r i d e a n d a c o n c o m i t a n t d e c r e a s e in s u l f a t e e x c r e t i o n o r a c h a n g e in i n t r a c e l l u l a r s u l f a t e levels. S u c h a s i t u a t i o n c o u l d l e a d to e i t h e r a d e p l e t i o n o f s e r u m s u l f a t e levels d u e to t i s s u e s e q u e s t r a t i o n of t h e ion o r a n e l e v a t i o n o f s e r u m s u l f a t e levels a s s o c i a t e d w i t h i m p a i r e d u p t a k e of s u l f a t e b y C F tissues. A l t e r n a t i v e l y , c o m p e n s a t o r y c h a n g e s in a b s o r p t i o n a n d e x c r e t i o n c o u l d p r o d u c e a s t a t e of n o r m o s u l f a t e m i a e v e n in t h e face of s e q u e s t r a t i o n of t h e a n i o n in s e c r e t o r y tissues a n d t h e e v e n t u a l loss of s u l f a t e via t h e release of h i g h l y - s u l f a t e d s e c r e t o r y p r o d u c t s . T h e r e s u l t s of t h e c u r r e n t s t u d y e x t e n d o u r k n o w l e d g e a b o u t the level of s u l f a t e in t h e s e r u m o f C F a n d n o n - C F p a t i e n t s , w i t h n o sexual or CF-related differences documented.
Acknowledgements T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m t h e C y s t i c F i b r o s i s F o u n d a t i o n . T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e t h e a s s i s t a n c e of Dr. R i c h a r d T h o m p s o n , D e p t . o f P u b l i c H e a l t h , U . A . B . in t h e p e r f o r m a n c e o f ion c h r o m a t o g r a p h y . Drs. R a y m o n d L y r e n e a n d R a l p h T i l l e r a n d Ms. M a r y B a u c o m o f the U . A . B . C y s t i c F i b r o s i s C e n t e r a n d Dr. W i l l i a m D a n i e l a n d Ms. A n n e T u r n e r a r e t h a n k e d for the c o l l e c t i o n o f sera f r o m C F a n d n o n - C F p a t i e n t s .
References I Boat TF, Cheng P. Cystic fibrosis. In: Mangos JA, Talamo RC, eds. New York: Stratton Intercontinental Medical Book Corp., 1976: 165-178. 2 Frates RC, Kaizu TT, Last JA. Mucus glycoproteins secreted by respirator3' epithelial tissue from cystic fibrosis patients. Pediat Res 1983; 17:30 34, 3 ho K. Kimata K, Sobue M, Suzuki S. Altered proteoglycan synthesis by epiphyseal cartilages m culture at low SO4- concentration. J Biol Chem 1982; 257:917 923 4 Sobue M, Takeuchi J, Ito K, Kimata K, Suzuki S. Effect of environmental sulfate concentrahons on the synthesis of low and high sulfated chondroitin sulfates by chick embryo cartilage I Biol Chem 1978: 253: 6190-6196. 5 Cole DEC, Striver CR. Microassay of inorganic sulfate in biological fluids by controlled flo,x amon chromatography. J Chromatogr 1981; 225: 359-367. 6 Anderson C. Ion chromatography: a new technique for clinical chemistry. Clin ('hem lt~7~< 22: 1424-1426. 7 Cole DEC, Scriver CR. Age-dependent serum sulfate levels in children and adolescents. Clin ('him Acta 1980; 107: 135-139.
247 8 Jennings ML, Passow 1|. Anion transport across the erythrocyte membrane, in situ proleolysis at band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate. Biochim Biophys Acta 1979; 554:498 519. 9 I,evmson C. Chloride and sulfate transport in Ehrlich ascites tumor cells: evidence for a common transport mechanism. J Cell Physiol 1978: 95:23 32. 10 Ramjeesingh M, Gaarn A, Rothslein A. The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red cell membrane. Biochim Biophys Acta 1980; 599:127 139.