Raafat
H.
Maghrabi,4
B.Sc.,
ABSTRACT
Carol
Alterations
determined
in pooled
treated
for
(PBH),
protein-bound
but
and
only
PCM,
PBF
returned
the unfractioned to normal with a1-globulin carbohydrate
sera and
of fractionated malnutrition13 Waslien,5
35 children and
acid
to control
moieties
Molar
ratios
as follows:
social
levels
with
the
PBSA>
Changes in serum glycoprotein levels with disease have been well-documented (l), but only recently have alterations in glycoproteins been reported with malnutrition. Patwardhan et al. (2) found increased levels of protein-bound hexose (PBH) and alterations in the ratios of the electrophoretic glycoprotein fractions in children with protein-calorie malnutrition (PCM) (2). The increase in PBI-I coincided with a decrease in total protein in these children, suggesting that the ratio of carbohydrate to protein in the glycoproteins was also disturbed. The electrophoretic technique used by these authors measured only total reducing sugars, and hence could not reveal whether the alteration had occurred in one or several carbohydrate moieties. In addition, without a suitable measurement of total glycoprotein one cannot determine whether changes occurred in the absolute amounts of any glycoprotein. In the current investigation the serum fractions were separated and isolated and their individual protein and carbohydrate contents were determined, making it possible to measure changes in the concentrations of each fraction as well as in its carbohydrate composition. Materials
and
methods
Five-milliliter blood samples were obtained from 35 children 6 to 36 months of age who were hospitalized with the classical symptoms of PCM, including growth failure, edema, and extreme apathy, and exhibiting total protein and albumin levels below 5.5 and 2.5 g/I00 ml
146
The American
Journal
of Clinical
treatment.
Nutrition
Ratios
but PBH change-in individual
protein
fractions
malnutrition controls.
protein-bound
PBH
of serum
protein-calorie
status-matched and
in PCM marked in
composition with
(PBSA),
serum were elevated treatment. The most
fraction.
Ph.D.
in the carbohydrate from
24 agesialic
I.
serum
fucose
Total
were
globulin
> PBF.
fractions Am.J.
Cliii.
carbohydrate
in PCM,
to protein
in
ratios returned occurred in the
indicated Nutr.
hexose
elevated
and PBF protein or globulin carbohydrate composition
been
30 children
protein-bound
(PBF)
of bound
have
(PCM),
an 29: 146
increase 150,
in 1976.
serum, respectively. Samples were also obtained from 30 other hospitalized children treated for PCM, whose total serum protein and albumin levels had attained normal limits. The condition and treatment of these children has been described in a previous study (3). A third group of 24 apparently healthy children matched for age and social status were chosen as controls. Pooled serum samples, each composed of equal amounts of sera from five children, were fractionated by preparative polyacrylamide electrophoresis using the apparatus described by Jovin et al. (4). In the first fractionation a gel column consisting of a 4.5-cm separating gel of 6% acrylamide and a 0.75-cm stacking gel of 2.5% acrylamide was prepared. A current of 15 ma was applied to this column for 1 hr to elute foreign ultraviolet-absorbing materials. The current was then discontinued and the pooled serum (approximately 10 ml) was layered onto the surface of the gel. A starting current of 15 ma was reapplied until the sample completely penetrated the stacking gel, as evidenced from the added bromphenol blue tracking dye. The current was then
1 From the Biochemistry Department, United States Medical Research Unit No. 3. c/o Spanish Embassy, Cairo, Egypt. 2 Supported by the Bureau of Medicine and Surgery, U.S. Navy Research Project MRO4I.20.Ol 0355, ONR Grant 90218, and Public Health Service Grant AM 08317 to Vanderbilt University, Nashville, Tennessee. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department, the naval service at large, or the Egyptian Ministry of Health. #{176} Research Assistant, U.S. Naval Medical Research Unit No. 3. Associate Professor, Auburn University, Auburn, Alabama: formerly Research Associate, Vanderbilt University School of Medicine and Head of Biochemistry Department. U.S. Naval Medical Research Unit No. 3.
29: FEBRUARY
1976,
pp.
146
150.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/2/146/4793349 by California Digital Library user on 01 January 2019
The bound carbohydrates proteins in protein-calorie
BOUND
CARBOHYDRATES
IN
PROTEIN-CALORIE
elevated
The data obtained from measurements of the total PBH, PBSA, and PBF contents of seven pooled sera from children with PCM, six from children recovering from PCM, and five from controls are shown in Table I. Control values for total PBH, PBF, and PBSA were similar to those previously reported for the same age range (2, 9, 10) and were not dissimilar from adult values (7, 11). TABLE
c arbohydrates
with
were
earlier
studies As in the previous study, there was no significant change in total PBH or PBSA during the treatment period, but PBF was significantly lower in the treated children. With the increase in total serum protein concentration after treatment of PCM, there was a drop in the total PBH and PBF to protein ratios to levels found in control children. However, the drop in PHSA to protein ratio, although marked, was not sufficient to restore a normal mean ratio. The ratios of the carbohydrates to globulin showed the same tendencies as those to total protein. The ratio of carbohydrate to protein in individual globulin fractions was higher in all of the globulin fractions in PCM (Table 2). With treatment the PBH to protein ratio was restored to control levels in each fraction. The PBF to protein ratios similarly decreased significantly. In fact, the PBF ratios were lower in treated PCM than in control children for each fraction. The PBSA to protein ratio for a-globulin was not significantly elevated in PCM, and although the ratio in both a1- and a2-globulin decreased significantly with treatment, it remained significantly higher than for controls in the a2-globulin fraction.
Overall
there
was
an
approxi-
150% increase in the total carboto protein ratio in the a1-globulin and a 50% increase in the ratios for a2-, and -y-globulin. The absolute levels of carbohydrate bound to each fraction, which can be calculated from the preceding carbohydrate to protein ratios
and
fraction the
the
absolute
(Table
majority
ratio
of
levels
3), indicate
a1-globulin
of unfrac tionated
se rum
the
of each
that
was
responsible
elevations
globulin
the change
in total
for
protein-
in PCM
PBH Subjects
mg/IOOml serum
PCM
118
TreatedPCM Controls
114± 96 ±
b
values
I
protein-bound
13.
PBSA, and PBF PCM, in agreement
in (2).
mately hydrate
Results
The
PBH,
Total
147
Those
values
with
different
±2#{176}
PBSA mg/lOOmI serum
g/IOOg protein 2.9
101
10#{176} 10#{176}
1.6 1.4
101±6#{176}
superscripts
are
68 significantly
±20#{176} ±
7#{176} different
PBF g/IOOg protein 2.5
mg/IOOml serum
g/IOOg protein
12.9±4.1#{176}
0.31
1.4
8.9±
1.0
8.9
(P < 0.05).
±
1.1”
0.12
1.0#{176}
0.13
of the
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/2/146/4793349 by California Digital Library user on 01 January 2019
increased to 45 ma for about 16 hr. at which time all protein had been eluted from the column. The downward migrating rings of resolved proteins were eluted at a flow rate of I ml/min using a finger pump and were monitored by continuous measurements of their ultraviolet absorption at 260 nm in a Beckman DB spectrophotometer. Three-milliliter fractions were collected using a fraction collector with a drop-counting attachment. The peaks of each eluted protein corresponding to the fractions found on cellulose acetate were combined after ascertaining the purity of the protein in the ascending and descending slopes by means of disc electrophoresis as described by Davis and Ornstein (5). This fractionation yielded peaks for albumin, a,globulin, and fl,-globulin, in addition to a large terminal peak consisting of fl2-globulin, a2-globulin, and the ‘y-globulins. The large terminal peak was then concentrated using a 2.5% acrylamide stacking gel and was subsequently layered onto a second 2-cm fractionation column consisting of 4.5% acrylamide. A flow rate and fraction volume of 0.75 ml/min and 2 ml, respectively, were used. Electrophoresis was completed in 10 hr, yielding separate peaks of fl2-globulin, a2-globulin, and the ‘y-globulins. The two fl-fractions were combined for subsequent analyses. The protein content of each protein fraction was determined by the optical density method of Warburg and Christian (6) and the carbohydrate contents of the protein were estimated as galactose and mannose (PBH), fucose (PBF), and sialic acid (PBSA) using the methods described by Winzler (7). Chemical and electrophoretic measurements were also performed on the unfractionated serum. The total protein concentration was determined by the biuret method of Weichselbaum (8). Serum total and glycoprotein electrophoresis were carried out on cellulose acetate membranes using the Beckman microzone electrophoretic system (model 101).
MALNUTRITION
MAGHRABI
148 TABLE
WASLIEN
2
Ratio of bound g carbohydrate/lOO
carbohydrate g protein-’
to protein
Subjects
levels
a,-Globulin
fl-Globulin
‘y-Globulin
5.9 ± 1.5#{176} 4.0±0.7#{176} 4.1±0.9#{176}
4.3 ± 1.3#{176} 3.0±0.5#{176} 3.0±0.8#{176}#{176}
1.9 ± 0.9#{176} 1.3 ±0.2#{176} 1.4 ±0.2#{176}
14.3 ± 3.5#{176} 7.1 ± 2.8#{176} 5.3±3.0#{176}
5.1 ± 1.0#{176} 3.6±0.5#{176} 2.7 ±0.7’
3.4 ± 1.2#{176} 2.7 ±0.3#{176} 2.2 ±0.6#{176}
1.6 ± 0.8#{176} 1.2 ±0.2#{176} 1.0±0.2#{176}
± ± ±
0.50#{176} 0.23#{176} 0.20#{176}
0.63 0.31 0.36
0.19#{176} 0.05#{176} 0.04#{176}
± ± ±
0.47 0.23 0.27
± ± ±
0.20”’ 0.03#{176} 0.04’
0.20 0.10 0.12
± ± ±
0.11#{176} 0.02#{176} 0.03#{176}’
100 g 33.4 15.9 13.1
Mean ± standard ‘ Those values
100 ml
fractions,
deviation. with different
of protein-bound
superscripts
carbohydrates
11.6 7.9 7.2
8.2 5.9 5.5
are significantly
in each
3.7 2.6 2.5
(P < 0.05).
different
globulin
serum#{176}
Subjects
Globulins PCM Treated PCM Controls PBH PCM Treated PCM Controls PBSA PCM Treated PCM Controls
a -Globulin mg r total
a,-Globulin
fl-Globulin
‘y-Globulin
mg
% total
mg
% total
30 30 26
280 640 740
12
8
720 1,080 810
40 24 17
33 20 17
42 43 33
36 38 32
33 20 13
32 20 18
37 39 22
36 38 32
230 290 240
10 8
% total
mg
23
1,130 1,630 1,380
48 45 44
12 19 22
lO 17 23
21 22 19
18 19 20
10 17 16
9 17 23
18 20 14
17 19 20
18
PBF PCM Treated
PCM
Controls
“Mg carbohydrate x g globulin/ 100 ml
4.3 1.8 1.5 in globulin unfractionated
33 20 16
4.5 3.3 2.9
fraction/ 100 ml serum serum.
bound carbohydrates. The a1-globulin fraction contributed 23 mg of the extra PBH to the total, whereas a2-globulin and -y-globulin contributed only 9 and 2 mg, respectively, and fl-globulin actually contributed 10 mg less than it did in the control sera. A similar pattern is observed for both PBSA and PBF, with a1-globulin contributing nearly one-third of the carbohydrate in each case, while in the controls it contributed only 16 to 18%.
34 37 32 =
1.3 1.4 1.9
g carbohydrate/
100 g protein
The approximate bohydrates was children
10 15 21
for
each
2.2 1.7 1.6 in isolated
molar similar globulin
17 19 17
globulin
fraction
ratio of the carwithin all groups of fraction,
but
was
altered with PCM and its treatment (Table 4). The molar ratios of PBH to PBF were lower in PCM (8.4 to 8.8) than in treated PCM (11.9 to 12.0) or controls (10.3 to 10.8), whereas PBSA to PBF ratios in PCM (3.9 to 4.3) were lower than in treated PCM (6.1 to 6.5) but similar to control ratios (4.0 to 4.5).
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/2/146/4793349 by California Digital Library user on 01 January 2019
Controls
fraction,!
globulin
17.2 ± 3.3#{176} 8.2±3.2#{176} 7.2±3.4#{176}
1.89 0.63 0.64
Total carbohydrate/ protein PCM Treated PCM Controls
TABLE 3 The absolute
in serum
a1-Globulin
Hexose PCM TreatedPCM Controls Sialic acid PCM TreatedPCM Controls Fucose PCM Treated PCM
a,
AND
BOUND TABLE The
CARBOHYDRATES
IN
PROTEIN-CALORIE
149
MALNUTRITION
4
molar
ratios
of carbohydrate
bound
to serum
globu
lin
fractions#{176} a2-Globulin
a -Globulin
$-G lob ulin
‘1-Glob
ulin
Subjects
PBSA
8.4 12.0 10.4
4.1 6.1 4.5
molar
= glucose, molecular weight molecular weight of 164.
The
approximate
PBSA PCM
were
(2.0
remained
not
to lower
Thus, absolute the total below
8.7 11.9 10.6
2.0 2.0 2.3
PCM
PBH
#{176}
fucose,
molar
ratios
changed
with
2.2
versus
than
for
1.8 controls
in each
of
180: PBSA
to
=
PBH
of
treatment
to of and
2.0),
(2.3
globulin
bles 2 and 3) with treatment PBH and PBSA absolute
to 2.6).
fraction
(Ta-
of PCM, levels were
while main-
tained plete
in the serum and showed return to normal in each
tion.
This
only globulin
incomfrac-
was reflected in a disproportionate in both PBH and PBSA over PBF in treated PCM, and in PBSA, which was
increase the present
PBF
in
higher
molar
ratios
than
either
or PBH.
Discussion The
observed
carbohydrates
in
increases
in
total
the
of
children
serum
bound with
PCM appear to result primarily from increases in the carbohydrate bound to a1globulin and to a lesser extent to that bound to a2-globulin. The values for total bound carbohydrates mained amount
PBI-I
PBSA
4.4 6.2 4.0
8.4 12.0 10.3
3.9 6.3 4.5
2.0 1.9 2.6
there was a reduction of relative and amounts of PBF to normal levels in serum (Table I) and to normal or normal
PBSA
PBH
PBSA
8.8 12.0 10.8
4.3 6.5 4.5
ratio of
PBH relative to PBSA PCM Treated Controls
-
ratios
Controls
Approximate
PBH
in above normal of a1-globulin
treated
PCM
primarily increased
cases because and its
rethe car-
bohydrate content did not fall completely to control levels. In contrast to the increased ratio of serum carbohydrate to protein observed in children with PCM, Weimer reported that rats subjected to protein depletion showed a significant decrease in the ratio of hexose to protein (12). Davis and Richmond (13) found no
2.2 1.9 2.3
N-acetylneuraminic
acid,
molecular
2.0 1.8 2.4 weight
of 309:
PBF
=
significant changes in the ratios of hexosamine and sialic acid to protein in rats fed protein-free diets and then repleted with diets containing 10 to 40% protein, and neither depleted nor repleted animals had ratios which differed from control levels. However, protein deficiency in rats differs in many respects from that observed in children, and only when extremely low levels of protein are fed, starting at an early age, is it possible to observe the edema and skin changes characterizing PCM (13). In addition, PCM is usually accompanied by frequent episodes of severe gastroenteritis and respiratory tract infections. Such infections and inflammations have been shown to cause elevations in PBH and other serum protein-bound carbohydrates (I), which has been thought to be mediated by endogenous humoral factors (14). However, the marked increase in a2- and fl-globulin which is observed in these infections was not observed in the cases of PCM. Perhaps the mechanisms responsible for addition of some carbohydrates to protein during infection have been maintained despite the decreased protein synthesis with protein deficiency. This may explain the disproportionate increases in sialic acid seen in PCM. It is also possible that liver damage, infection, and parasitic infestation, which are common in PCM, could result in reduced
synthesis
of
particular
glycoproteins
which are low in total carbohydrate a high proportion of sialic acid creased synthesis of a1-glycoglobulins in this carbohydrate moiety.
and have or in inhigh
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/2/146/4793349 by California Digital Library user on 01 January 2019
Approximate molar relative to PBF PCM Treated PCM
PBH
ISO
MAGHRABI
AND 8.
References I.
SHETL.AR,
origin 1961.
WASLIEN
M. R. The and significance.
serum glycoproteins, Ann. N.Y. Acad. Sci.
V.
Serum
vitamin
A,
retinol-binding
protein, and prealbumin concentrations in proteincalorie malnutrition. I. A functional defect in hepatic retinol release. Am. J. Clin. Nutr. 26: 973, 1973. 4. JovIN, T., A. CHRAMBRACH ANt) H. A. NAUGHTON. Preparative polyacrylamide electrophoresis. Anal. Biochem. 9: 351, 1964. 5. DAvIS, B. J., AND L. ORNSTEIN. Gel electrophoresis. Ann. N.Y. Acad. Sci. 121: 305. 6. WARBURG, 0., ANt) W. CHRISTIAN. Isolierung und Kristallisation des Garungsferments Enolase. Biochem. Z. 310: 384, 1941. 7. WINZLER, R. J. Determination of serum glycoproteins. In: Methods of Biochemical Analysis, New York: Wiley (Interscience), 1955, vol. II, p. 279.
9.
10. II.
COCKERELL,
G. L., AND W. R. BEISEL. Comparison of serum protein-bound carbohydrate and glycoprotein patterns of man, monkey, and rat. Brit. J. Exptl. Pathol. 54: 49, 1973.
12.
WEIMER,
13.
DAVIS,
H. E. The effects of protein depletion and repletion on the concentration and distribution of serum proteins and protein-bound carbohydrates of the adult rat. Ann. N.Y. Acad. Sci. 94: 225, 1961. M. M., AND J. dietary protein on serum 215: 366, 1968.
E. RICHMOND. proteins. Am.
Effect of J. Physiol.
14. COCKERELL, G. L. Changes in plasma protein-bound carbohydrates and glycoprotein patterns during infection, inflammation, and starvation. Proc. Soc. Exptl. Biol. Med. 142: 1072, 1973.
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/2/146/4793349 by California Digital Library user on 01 January 2019
PATWARDHAN.
method amounts Pathol.
their 94: 44,
N., R. H. MAGHRABI, W. MousA, M. K. GABR AND S. EL MARAGHY. Serum glycoproteins in protein-calorie deficiency disease. Am. J. Clin. Nutr. 24: 906, 1971. 3. SMrrLl, F. R., DEW. S. GOODMAN, M. S. ZAKLAMA, M. K. GABR, S. EL MARAGHY AND V. N.
2. PATWARDHAN,
T. E. An accurate and rapid for the determination of proteins in small of blood serum and plasma. Am. J. Clin. (Tech. Sect.) 16: 40, 1946. METWALLI, 0. M., Y. K. W. ABOUL DAHAB, A. S. KHALIFA AND A. G. WISHAHI. Variation of serum mucoproteins and seromucoids with age in Egyptian infants and children. J. Egypt. Med. Assoc. 52: 386, 1962. WEST, C. D., AND R. HONG. The glycoproteins of serum. J. Pediat. 60: 430, 1962. WEICLISEI.BAUM,
H.
Maghrabi,4
B.Sc.,
ABSTRACT
Carol
Alterations
determined
in pooled
treated
for
(PBH),
protein-bound
but
and
only
PCM,
PBF
returned
the unfractioned to normal with a1-globulin carbohydrate
sera and
of fractionated malnutrition13 Waslien,5
35 children and
acid
to control
moieties
Molar
ratios
as follows:
social
levels
with
the
PBSA>
Changes in serum glycoprotein levels with disease have been well-documented (l), but only recently have alterations in glycoproteins been reported with malnutrition. Patwardhan et al. (2) found increased levels of protein-bound hexose (PBH) and alterations in the ratios of the electrophoretic glycoprotein fractions in children with protein-calorie malnutrition (PCM) (2). The increase in PBI-I coincided with a decrease in total protein in these children, suggesting that the ratio of carbohydrate to protein in the glycoproteins was also disturbed. The electrophoretic technique used by these authors measured only total reducing sugars, and hence could not reveal whether the alteration had occurred in one or several carbohydrate moieties. In addition, without a suitable measurement of total glycoprotein one cannot determine whether changes occurred in the absolute amounts of any glycoprotein. In the current investigation the serum fractions were separated and isolated and their individual protein and carbohydrate contents were determined, making it possible to measure changes in the concentrations of each fraction as well as in its carbohydrate composition. Materials
and
methods
Five-milliliter blood samples were obtained from 35 children 6 to 36 months of age who were hospitalized with the classical symptoms of PCM, including growth failure, edema, and extreme apathy, and exhibiting total protein and albumin levels below 5.5 and 2.5 g/I00 ml
146
The American
Journal
of Clinical
treatment.
Nutrition
Ratios
but PBH change-in individual
protein
fractions
malnutrition controls.
protein-bound
PBH
of serum
protein-calorie
status-matched and
in PCM marked in
composition with
(PBSA),
serum were elevated treatment. The most
fraction.
Ph.D.
in the carbohydrate from
24 agesialic
I.
serum
fucose
Total
were
globulin
> PBF.
fractions Am.J.
Cliii.
carbohydrate
in PCM,
to protein
in
ratios returned occurred in the
indicated Nutr.
hexose
elevated
and PBF protein or globulin carbohydrate composition
been
30 children
protein-bound
(PBF)
of bound
have
(PCM),
an 29: 146
increase 150,
in 1976.
serum, respectively. Samples were also obtained from 30 other hospitalized children treated for PCM, whose total serum protein and albumin levels had attained normal limits. The condition and treatment of these children has been described in a previous study (3). A third group of 24 apparently healthy children matched for age and social status were chosen as controls. Pooled serum samples, each composed of equal amounts of sera from five children, were fractionated by preparative polyacrylamide electrophoresis using the apparatus described by Jovin et al. (4). In the first fractionation a gel column consisting of a 4.5-cm separating gel of 6% acrylamide and a 0.75-cm stacking gel of 2.5% acrylamide was prepared. A current of 15 ma was applied to this column for 1 hr to elute foreign ultraviolet-absorbing materials. The current was then discontinued and the pooled serum (approximately 10 ml) was layered onto the surface of the gel. A starting current of 15 ma was reapplied until the sample completely penetrated the stacking gel, as evidenced from the added bromphenol blue tracking dye. The current was then
1 From the Biochemistry Department, United States Medical Research Unit No. 3. c/o Spanish Embassy, Cairo, Egypt. 2 Supported by the Bureau of Medicine and Surgery, U.S. Navy Research Project MRO4I.20.Ol 0355, ONR Grant 90218, and Public Health Service Grant AM 08317 to Vanderbilt University, Nashville, Tennessee. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department, the naval service at large, or the Egyptian Ministry of Health. #{176} Research Assistant, U.S. Naval Medical Research Unit No. 3. Associate Professor, Auburn University, Auburn, Alabama: formerly Research Associate, Vanderbilt University School of Medicine and Head of Biochemistry Department. U.S. Naval Medical Research Unit No. 3.
29: FEBRUARY
1976,
pp.
146
150.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/2/146/4793349 by California Digital Library user on 01 January 2019
The bound carbohydrates proteins in protein-calorie
BOUND
CARBOHYDRATES
IN
PROTEIN-CALORIE
elevated
The data obtained from measurements of the total PBH, PBSA, and PBF contents of seven pooled sera from children with PCM, six from children recovering from PCM, and five from controls are shown in Table I. Control values for total PBH, PBF, and PBSA were similar to those previously reported for the same age range (2, 9, 10) and were not dissimilar from adult values (7, 11). TABLE
c arbohydrates
with
were
earlier
studies As in the previous study, there was no significant change in total PBH or PBSA during the treatment period, but PBF was significantly lower in the treated children. With the increase in total serum protein concentration after treatment of PCM, there was a drop in the total PBH and PBF to protein ratios to levels found in control children. However, the drop in PHSA to protein ratio, although marked, was not sufficient to restore a normal mean ratio. The ratios of the carbohydrates to globulin showed the same tendencies as those to total protein. The ratio of carbohydrate to protein in individual globulin fractions was higher in all of the globulin fractions in PCM (Table 2). With treatment the PBH to protein ratio was restored to control levels in each fraction. The PBF to protein ratios similarly decreased significantly. In fact, the PBF ratios were lower in treated PCM than in control children for each fraction. The PBSA to protein ratio for a-globulin was not significantly elevated in PCM, and although the ratio in both a1- and a2-globulin decreased significantly with treatment, it remained significantly higher than for controls in the a2-globulin fraction.
Overall
there
was
an
approxi-
150% increase in the total carboto protein ratio in the a1-globulin and a 50% increase in the ratios for a2-, and -y-globulin. The absolute levels of carbohydrate bound to each fraction, which can be calculated from the preceding carbohydrate to protein ratios
and
fraction the
the
absolute
(Table
majority
ratio
of
levels
3), indicate
a1-globulin
of unfrac tionated
se rum
the
of each
that
was
responsible
elevations
globulin
the change
in total
for
protein-
in PCM
PBH Subjects
mg/IOOml serum
PCM
118
TreatedPCM Controls
114± 96 ±
b
values
I
protein-bound
13.
PBSA, and PBF PCM, in agreement
in (2).
mately hydrate
Results
The
PBH,
Total
147
Those
values
with
different
±2#{176}
PBSA mg/lOOmI serum
g/IOOg protein 2.9
101
10#{176} 10#{176}
1.6 1.4
101±6#{176}
superscripts
are
68 significantly
±20#{176} ±
7#{176} different
PBF g/IOOg protein 2.5
mg/IOOml serum
g/IOOg protein
12.9±4.1#{176}
0.31
1.4
8.9±
1.0
8.9
(P < 0.05).
±
1.1”
0.12
1.0#{176}
0.13
of the
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/2/146/4793349 by California Digital Library user on 01 January 2019
increased to 45 ma for about 16 hr. at which time all protein had been eluted from the column. The downward migrating rings of resolved proteins were eluted at a flow rate of I ml/min using a finger pump and were monitored by continuous measurements of their ultraviolet absorption at 260 nm in a Beckman DB spectrophotometer. Three-milliliter fractions were collected using a fraction collector with a drop-counting attachment. The peaks of each eluted protein corresponding to the fractions found on cellulose acetate were combined after ascertaining the purity of the protein in the ascending and descending slopes by means of disc electrophoresis as described by Davis and Ornstein (5). This fractionation yielded peaks for albumin, a,globulin, and fl,-globulin, in addition to a large terminal peak consisting of fl2-globulin, a2-globulin, and the ‘y-globulins. The large terminal peak was then concentrated using a 2.5% acrylamide stacking gel and was subsequently layered onto a second 2-cm fractionation column consisting of 4.5% acrylamide. A flow rate and fraction volume of 0.75 ml/min and 2 ml, respectively, were used. Electrophoresis was completed in 10 hr, yielding separate peaks of fl2-globulin, a2-globulin, and the ‘y-globulins. The two fl-fractions were combined for subsequent analyses. The protein content of each protein fraction was determined by the optical density method of Warburg and Christian (6) and the carbohydrate contents of the protein were estimated as galactose and mannose (PBH), fucose (PBF), and sialic acid (PBSA) using the methods described by Winzler (7). Chemical and electrophoretic measurements were also performed on the unfractionated serum. The total protein concentration was determined by the biuret method of Weichselbaum (8). Serum total and glycoprotein electrophoresis were carried out on cellulose acetate membranes using the Beckman microzone electrophoretic system (model 101).
MALNUTRITION
MAGHRABI
148 TABLE
WASLIEN
2
Ratio of bound g carbohydrate/lOO
carbohydrate g protein-’
to protein
Subjects
levels
a,-Globulin
fl-Globulin
‘y-Globulin
5.9 ± 1.5#{176} 4.0±0.7#{176} 4.1±0.9#{176}
4.3 ± 1.3#{176} 3.0±0.5#{176} 3.0±0.8#{176}#{176}
1.9 ± 0.9#{176} 1.3 ±0.2#{176} 1.4 ±0.2#{176}
14.3 ± 3.5#{176} 7.1 ± 2.8#{176} 5.3±3.0#{176}
5.1 ± 1.0#{176} 3.6±0.5#{176} 2.7 ±0.7’
3.4 ± 1.2#{176} 2.7 ±0.3#{176} 2.2 ±0.6#{176}
1.6 ± 0.8#{176} 1.2 ±0.2#{176} 1.0±0.2#{176}
± ± ±
0.50#{176} 0.23#{176} 0.20#{176}
0.63 0.31 0.36
0.19#{176} 0.05#{176} 0.04#{176}
± ± ±
0.47 0.23 0.27
± ± ±
0.20”’ 0.03#{176} 0.04’
0.20 0.10 0.12
± ± ±
0.11#{176} 0.02#{176} 0.03#{176}’
100 g 33.4 15.9 13.1
Mean ± standard ‘ Those values
100 ml
fractions,
deviation. with different
of protein-bound
superscripts
carbohydrates
11.6 7.9 7.2
8.2 5.9 5.5
are significantly
in each
3.7 2.6 2.5
(P < 0.05).
different
globulin
serum#{176}
Subjects
Globulins PCM Treated PCM Controls PBH PCM Treated PCM Controls PBSA PCM Treated PCM Controls
a -Globulin mg r total
a,-Globulin
fl-Globulin
‘y-Globulin
mg
% total
mg
% total
30 30 26
280 640 740
12
8
720 1,080 810
40 24 17
33 20 17
42 43 33
36 38 32
33 20 13
32 20 18
37 39 22
36 38 32
230 290 240
10 8
% total
mg
23
1,130 1,630 1,380
48 45 44
12 19 22
lO 17 23
21 22 19
18 19 20
10 17 16
9 17 23
18 20 14
17 19 20
18
PBF PCM Treated
PCM
Controls
“Mg carbohydrate x g globulin/ 100 ml
4.3 1.8 1.5 in globulin unfractionated
33 20 16
4.5 3.3 2.9
fraction/ 100 ml serum serum.
bound carbohydrates. The a1-globulin fraction contributed 23 mg of the extra PBH to the total, whereas a2-globulin and -y-globulin contributed only 9 and 2 mg, respectively, and fl-globulin actually contributed 10 mg less than it did in the control sera. A similar pattern is observed for both PBSA and PBF, with a1-globulin contributing nearly one-third of the carbohydrate in each case, while in the controls it contributed only 16 to 18%.
34 37 32 =
1.3 1.4 1.9
g carbohydrate/
100 g protein
The approximate bohydrates was children
10 15 21
for
each
2.2 1.7 1.6 in isolated
molar similar globulin
17 19 17
globulin
fraction
ratio of the carwithin all groups of fraction,
but
was
altered with PCM and its treatment (Table 4). The molar ratios of PBH to PBF were lower in PCM (8.4 to 8.8) than in treated PCM (11.9 to 12.0) or controls (10.3 to 10.8), whereas PBSA to PBF ratios in PCM (3.9 to 4.3) were lower than in treated PCM (6.1 to 6.5) but similar to control ratios (4.0 to 4.5).
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Controls
fraction,!
globulin
17.2 ± 3.3#{176} 8.2±3.2#{176} 7.2±3.4#{176}
1.89 0.63 0.64
Total carbohydrate/ protein PCM Treated PCM Controls
TABLE 3 The absolute
in serum
a1-Globulin
Hexose PCM TreatedPCM Controls Sialic acid PCM TreatedPCM Controls Fucose PCM Treated PCM
a,
AND
BOUND TABLE The
CARBOHYDRATES
IN
PROTEIN-CALORIE
149
MALNUTRITION
4
molar
ratios
of carbohydrate
bound
to serum
globu
lin
fractions#{176} a2-Globulin
a -Globulin
$-G lob ulin
‘1-Glob
ulin
Subjects
PBSA
8.4 12.0 10.4
4.1 6.1 4.5
molar
= glucose, molecular weight molecular weight of 164.
The
approximate
PBSA PCM
were
(2.0
remained
not
to lower
Thus, absolute the total below
8.7 11.9 10.6
2.0 2.0 2.3
PCM
PBH
#{176}
fucose,
molar
ratios
changed
with
2.2
versus
than
for
1.8 controls
in each
of
180: PBSA
to
=
PBH
of
treatment
to of and
2.0),
(2.3
globulin
bles 2 and 3) with treatment PBH and PBSA absolute
to 2.6).
fraction
(Ta-
of PCM, levels were
while main-
tained plete
in the serum and showed return to normal in each
tion.
This
only globulin
incomfrac-
was reflected in a disproportionate in both PBH and PBSA over PBF in treated PCM, and in PBSA, which was
increase the present
PBF
in
higher
molar
ratios
than
either
or PBH.
Discussion The
observed
carbohydrates
in
increases
in
total
the
of
children
serum
bound with
PCM appear to result primarily from increases in the carbohydrate bound to a1globulin and to a lesser extent to that bound to a2-globulin. The values for total bound carbohydrates mained amount
PBI-I
PBSA
4.4 6.2 4.0
8.4 12.0 10.3
3.9 6.3 4.5
2.0 1.9 2.6
there was a reduction of relative and amounts of PBF to normal levels in serum (Table I) and to normal or normal
PBSA
PBH
PBSA
8.8 12.0 10.8
4.3 6.5 4.5
ratio of
PBH relative to PBSA PCM Treated Controls
-
ratios
Controls
Approximate
PBH
in above normal of a1-globulin
treated
PCM
primarily increased
cases because and its
rethe car-
bohydrate content did not fall completely to control levels. In contrast to the increased ratio of serum carbohydrate to protein observed in children with PCM, Weimer reported that rats subjected to protein depletion showed a significant decrease in the ratio of hexose to protein (12). Davis and Richmond (13) found no
2.2 1.9 2.3
N-acetylneuraminic
acid,
molecular
2.0 1.8 2.4 weight
of 309:
PBF
=
significant changes in the ratios of hexosamine and sialic acid to protein in rats fed protein-free diets and then repleted with diets containing 10 to 40% protein, and neither depleted nor repleted animals had ratios which differed from control levels. However, protein deficiency in rats differs in many respects from that observed in children, and only when extremely low levels of protein are fed, starting at an early age, is it possible to observe the edema and skin changes characterizing PCM (13). In addition, PCM is usually accompanied by frequent episodes of severe gastroenteritis and respiratory tract infections. Such infections and inflammations have been shown to cause elevations in PBH and other serum protein-bound carbohydrates (I), which has been thought to be mediated by endogenous humoral factors (14). However, the marked increase in a2- and fl-globulin which is observed in these infections was not observed in the cases of PCM. Perhaps the mechanisms responsible for addition of some carbohydrates to protein during infection have been maintained despite the decreased protein synthesis with protein deficiency. This may explain the disproportionate increases in sialic acid seen in PCM. It is also possible that liver damage, infection, and parasitic infestation, which are common in PCM, could result in reduced
synthesis
of
particular
glycoproteins
which are low in total carbohydrate a high proportion of sialic acid creased synthesis of a1-glycoglobulins in this carbohydrate moiety.
and have or in inhigh
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Approximate molar relative to PBF PCM Treated PCM
PBH
ISO
MAGHRABI
AND 8.
References I.
SHETL.AR,
origin 1961.
WASLIEN
M. R. The and significance.
serum glycoproteins, Ann. N.Y. Acad. Sci.
V.
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COCKERELL,
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12.
WEIMER,
13.
DAVIS,
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