*Third Department of Internal Medicine, Gunma University School of
Medicine, Maebashi, Gunma, Japan. Shirane Clinic, Numata, Gunma, Japan. Nishikatagai Clinic, Maebashi; Gunma, Japan. §Wakaba Hospital, Maebashi, Gunma, Japan.
Correspondence to Dr. Yoshihisa Nojima, Third Department of Internal Medicine,
Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan. Phone:
+81 27 220 8161; Fax: +81 27 220 8173; E-mail:
ynojima{at}news.sb.gunma-u.ac.jp
Abstract. Sulfite, a well known air pollutant, is toxic for
humans,especially those with sulfite hypersensitivity. Sulfite is also
generatedendogenously, during normal metabolism of sulfur-containingamino
acids. Mammalian tissues contain the enzyme sulfite oxidase,which detoxifies
both endogenous and exogenous sulfite by oxidationto sulfate. Deficiency of
sulfite oxidase in humans is fatal,demonstrating its physiologic importance.
Nevertheless, informationabout serum and tissue levels of sulfite in normal
and pathologicconditions is limited. Using a sensitive HPLC assay, it is
shownhere that sera from patients with chronic renal failure (CRF)contain
significantly higher amounts of sulfite than those fromhealthy subjects. Mean
± SD of serum sulfite in healthysubjects (n = 20) was 1.55
± 0.54 µM, whereas thosein patients under maintenance hemodialysis
(HD patients; n =44) and CRF patients before introducing dialysis
therapy (pre-HDpatients; n = 33) were 3.23 ± 1.02 µM
(P < 0.01)and 3.80 ± 3.32 µM (P < 0.01),
respectively.Among pre-HD patients, serum sulfite was positively correlated
withserum creatinine (r = 0.714, P < 0.0001), and
negativelywith serum albumin (r = -0.407, P = 0.0188),
hematocrit (r =-0.524, P = 0.0017), and total cholesterol
(r = -0.375, P =0.0318). There was no significant
association between sulfiteand patient age, gender, or leukocyte counts.
Multiple regressionanalysis revealed serum creatinine as the sole independent
predictorof serum sulfite levels. Each HD treatment was associated with
approximately27% reduction in serum sulfite levels, suggesting the presence
ofa dialyzable form in serum. Thus, these results indicate thatreduced
glomerular filtration is a factor that determines serumsulfite levels.
Chronic elevation in serum sulfite levels mightcontribute to tissue or organ
dysfunction in patients with CRF.
Sulfur dioxide is an air pollutant released into the atmosphereduring the
combustion of fossil fuel
(1,2).
Sulfur dioxide canbe converted to sulfite upon contact with fluids lining the
airpassages. Sulfite and its related compounds, such as metabisulfite,are
also widely used in food preservation as antimicrobial agentsand antioxidants
(3). In addition, endogenous
sulfite is generatedduring the normal metabolic processing of
sulfur-containingamino acids or drugs
(4,5).
The toxic effects of sulfite onmammals have been studied extensively
(6,7,8,9,10,11).
It cancause allergic reactions in humans; most commonly, bronchoconstriction
inasthmatics (6). Mammalian
tissues contain sulfite oxidase, whichcatalyzes the oxidative detoxification
of sulfite (12). Childrenwith
hereditary deficiency in this enzyme develop mental retardation,neurologic
symptoms such as spastic quadriplegia, and earlydeath
(4,13,14).
Thus, sulfite levels in the body must normallybe tightly regulated.
We and others have recently demonstrated that human or rabbitneutrophils
produce sulfite spontaneously or in response tostimulation with the bacterial
endotoxin lipopolysaccharide
(15,16,17).
Wealso found that in vivo administration of lipopolysaccharideinto
rats induced a significant increase in serum sulfite concentration
(15).These results strongly
suggest that sulfite is not only an exogenoustoxic substance and an
endogenous metabolite, but also actsas a mediator of neutrophil function with
antimicrobial andproinflammatory activities. However, the effects of sulfite
oncellular functions are unclear. Moreover, little is known aboutthe role of
sulfite in various pathophysiologic conditions.
In the present study, we determined sulfite concentrations insera from
patients with chronic renal failure (CRF) using reversed-phaseHPLC, a
sensitive assay for serum sulfite recently developedby Ji et al.
(18). Compared with healthy
subjects, CRF patientshad significantly higher levels of serum sulfite. A
positivecorrelation between serum sulfite and creatinine among predialysis
CRFpatients demonstrates that reduced renal function is a contributingfactor
for elevated serum sulfite. Hemodialysis (HD) treatmentwas associated with a
temporary reduction of serum sulfite,suggesting that at least a portion of
serum sulfite is in adialyzable form.
Patients
This study was performed on healthy volunteers (n = 20), patients
withCRF not receiving dialysis (pre-HD patients; n = 33), and
patientsundergoing maintenance HD (HD patients; n = 44). HD was
performedthree times a week (12 to 15 h per week) using bicarbonate
dialysate.Half of our patients (n = 22) were dialyzed with cellulose
triacetatemembranes, and the others with polysulfone membranes (n =
6),vitamin E-modified membranes (n = 10), or miscellaneous
(n =6). Median duration ± SD of HD treatment was 43.5
±42.4 mo (0.5 to 254 mo). Patient profiles are summarized in
Table 1.As shown, the age and
gender of the three groups were comparable,except for the mean age of HD
patients, which was higher thanthose of control and pre-HD subjects
(P < 0.05). Patientsshowing evidence of intercurrent infection
and those takingantibiotics were excluded from the study. All patients gave
informedconsent. To assess dietary protein intake, the protein catabolicrate
(PCR) was calculated for HD patients according to the methoddescribed
previously (19).
Table 1. Clinical characteristics of the subjectsa
Sample Preparation
Blood samples were drawn from the antecubital veins of healthysubjects and
CRF patients and from the arterial side of thearteriovenous fistula of HD
patients before and after HD. Allpatients were fasted overnight before blood
collection. Afterseparation, serum was immediately subjected to sample
preparationas described previously
(15,18).
In brief, serum samples (100µl) were mixed with 70 µl of 0.212 M sodium
borohydridein 0.05 M Tris-HCl (pH 8.5) and incubated at room temperaturefor
30 min. This procedure is critical for the reductive releaseof protein-bound
sulfite, and sodium borohydride was freshlyprepared in each experiment.
Preliminary experiments revealedthat sulfite release was dependent on the
incubation periodwith borohydride and reached a plateau level in 30 min. The
sampleswere then mixed with 10 µl of 70 mM monobromobimane in
acetonitrile.After incubation for 10 min at 42°C, 50 µl of 1.5M
perchloric acid solution was added to the mixture followedby vortex mixing.
The protein precipitates were removed by centrifugationat 12,400 x
g for 10 min at room temperature. The supernatantwas immediately
neutralized by adding 10 µl of 2 M Tris,gently mixed, and centrifuged
again at 12,400 x g for 10 min.Ten microliters of the
neutralized supernatant was injectedonto HPLC column (Hitachi 655-A11
system), as described below.All procedures of sample preparation were
completed within 2h after serum separation.
Determination of Serum Sulfite Concentration using Reversed-Phase
HPLC with Fluorescence Detection
Samples were resolved on 4 x 250 mm C8 reversed-phase column(5-µm
packing; GL Science, Tokyo, Japan). The column wasequilibrated with
methanol:acetic acid:water (5.00:0.25:94.75,by volume, pH 3.4) and developed
with a gradient of methanolin acetic acid: water (0.25:94.5, by volume) at a
flow rateof 0.8 ml/min as follows: 0 to 5 min, 30 ml/L; 5 to 13 min,30 to
350 ml/L; 13 to 23 min, 350 to 620 ml/L; 23 to 24 min,620 to 1000 ml/L; 24 to
29 min, 1000 ml/L; 29 to 30 min, 1000to 30 ml/L; and 30 to 34 min, 30 ml/L.
Sulfitebimane was detectedby excitation at 390 nm and emission at 472 nm with
use of acutoff filter and eluted at 45 ml/L of methanol. Standard solutions
ofsulfite were prepared fresh for each assay by dissolving sodiumsulfite in
Hanks' balanced salt solution, and a calibrationcurve was obtained by
measuring relative fluorescence intensityof sulfite-bimane as described
previously (15). Linearity was
obtainedover a concentration range of sulfite from 0.12 to 125 µM.Each
serum sample was assayed in duplicate. The intra- and interassaycoefficients
of variation were 2.7 and 10.5%, respectively.
Statistical Analyses
Statistical analyses were performed using commercially availablepersonal
computer software, StatView 4.5. Data are presentedas mean ± SD.
Differences between mean values in studygroups were evaluated by the
t test. Correlation between twovariables was examined by simple
regression analysis. Independentassociations between one dependent and two or
more independentvariables were assessed by multiple regression analysis. The
differencewas considered significant at P < 0.05.
Ji et al. recently established a sensitive method for measuring
sulfiteconcentration in biologic fluids
(18). Using this method, they
showedthat normal human serum contained sulfite at a concentrationof 4.87
± 2.49 µM (mean ± SD). We also examinedserum sulfite levels
in the healthy Japanese population. Themean ± SD of serum sulfite in
20 Japanese subjects was1.55 ± 0.54 µM, which is considerably lower
thanthat determined by Ji. There was no significant correlationbetween serum
sulfite levels and subjects' age (r = 0.04, P= 0.867).
We next measured sulfite in sera from 77 patients with CRF.Forty-four
patients were undergoing maintenance HD therapy (HDpatients). The remaining
33 did not yet require dialysis (pre-HDpatients) and had serum creatinine
levels ranging from 1.5 to13.3 mg/dl (mean 5.47 ± 3.55). Compared with
healthycontrol subjects (Figure
1), both HD and pre-HD patients hadsignificantly higher levels of
serum sulfite (P < 0.01).The means (±SD) in HD and pre-HD
patients were 3.23 ±1.02 and 3.80 ± 3.32 µM, respectively.
There wasno significant difference in serum sulfite concentration between
pre-HDand HD patients.
Figure 1. Comparison of serum sulfite levels in sera of healthy subjects,
pre-hemodialysis (HD) patients, and HD patients. Horizontal lines at the top,
middle, and bottom of the boxes show the 75th, 50th, and 25th percentiles,
respectively, and vertical lines above and below the boxes show the 90th and
10th percentiles, respectively. Statistical analysis was performed by
t test.
As shown in Figure 1, the
level of serum sulfite was widelydistributed in pre-HD patients, ranging from
0.70 to 13.5 µM.Therefore, we attempted to determine which factors
contributeserum sulfite levels among pre-HD patients.
Table 2 gives asimple
correlation between serum sulfite and other variables.Serum sulfite levels
correlated positively with serum creatinineconcentration
(Figure 2A) (P <
0.0001), and inversely withserum albumin (P = 0.0188), total
cholesterol (P = 0.0318),and hematocrit
(Figure 2B) (P =
0.0017). Sulfite showed no significantassociation with age, gender, serum
total protein levels, orleukocyte counts. Multiple regression analysis was
also performedto evaluate independent factors affecting serum sulfite levels
(Table 3).In this analysis,
only the serum creatinine levels contributedsignificantly to the variability
of sulfite levels. Mean ±SD of PCR in 31 HD patients was 0.89 ±
0.16 g/kg perd. There was no significant correlation between PCR and serum
sulfitelevels among these patients (r = 0.099, P =
0.5945).
Figure 2. Correlation between serum sulfite levels and serum creatinine (A) and
between serum sulfite levels and hematocrit (B) in pre-HD patients. The
equations of linear regression are sulfite (mM) = 0.146 + 0.668 x
creatinine (mg/dl) (A) and sulfite (mM) = 11.612 - 0.252 x hematocrit
(%) (B).
Table 3. Multiple regression analysis of factors affecting serum sulfite levels
in pre-HD patientsa
We finally examined serum sulfite levels before and after HDtreatment in
10 HD patients. Each HD for these patients wasperformed using the cellulose
triacetate membrane. As shownin Figure
3, HD treatment led to the significant reduction (P<
0.01) in serum sulfite levels by an average of 27% (95%confidence interval,
17.7 to 33.9%). This suggests that serumsulfite is dialyzable to some
extent.
Figure 3. Changes in serum sulfite levels after HD treatment in 10 patients receiving
maintenance HD. Statistical analysis was performed by paired t
test.
In the current study, we measured serum concentration of sulfitein healthy
Japanese subjects and patients with CRF accordingto the method developed by
Ji et al. (18). When
compared withhealthy subjects, patients with CRF had a significantly higher
serumsulfite concentration. Abnormally high levels in serum sulfitehave been
demonstrated in patients with congenital deficiencyin sulfite oxidase and in
patients with hypersensitivity tosulfite
(4,20).
Our present study indicates that the elevationof serum sulfite levels is not
restricted to these uncommonpathologic conditions.
In our hands, sera from healthy control subjects contained sulfiteat a
concentration of 0.1 to 2.2 µM. Although levels ineach individual were
within the normal reference range (0 toapproximately 9.85 µM) determined
by Ji et al. (18),
themean value in our study was approximately 30% of that reportedby Ji
et al. Togawa et al.
(21) reported a far lower
value (0.47± 0.25 µM) as the mean concentration of serum sulfitein
healthy Japanese subjects, although their assay system wascompletely
different from ours. Possible explanations for thedisparity in normal
reference ranges between our study and thatof Ji include methodologic,
ethnic, dietary, and other environmentaldifferences. Although dietary intake
is certainly a source ofserum sulfite
(18), it is unlikely that
Japanese food and beveragescontain less sulfite as a preservative. Moreover,
among HD patients,PCR was not significantly correlated with serum sulfite
levels(r = 0.099, P = 0.5945), suggesting that dietary
protein intakecontributes little, if any, to the variation in serum sulfite
levelsamong these patients. One possible and likely cause could liein the
effectiveness of the borohydride treatment to reductivelyrelease serum
protein-bound sulfite. Because borohydride isrelatively unstable at pH 8.5,
incomplete release of sulfitecould account for the difference. At present,
however, it isunclear which of these factors is most important.
Among pre-HD patients, the level of serum sulfite was well correlatedwith
that of serum creatinine. Thus, the reduced glomerularfiltration may be a
cause of elevated serum sulfite concentration.Indeed, sulfate, an oxidative
product of sulfite, accumulatesin the serum and contributes to acidemia in
patients with end-stagerenal failure
(22). We also reported that
sulfite productionby activated neutrophils was dependent on the sulfate
concentrationin culture medium
(15). Hence, the higher serum
concentrationof sulfate in CRF patients may shift the equilibrium between
sulfiteand sulfate toward sulfite. It is also possible that enzymeactivity
of sulfite oxidase is impaired in CRF patients. Inaddition, metabolism of
sulfur-containing amino acids such asmethionine, cysteine, and homocysteine
has been reported tobe aberrant in CRF patients
(23,24).
Because sulfite is an intermediarymetabolite in the normal processing of
these amino acids
(4,5),
thismay be an alternative mechanism by which serum sulfite is elevatedin CRF
patients.
In our current protocol, sulfite was measured as total serumsulfite which
contains both free and protein-bound forms
(18).Because sulfite easily
reacts with disulfide bonds of proteinsand small molecules such as cysteine,
the half-life of freesulfite in healthy individuals is thought to be short.
At present,however, it remains unknown how much free sulfite is containedin
sera in healthy subjects and CRF patients. There was approximatelya 27%
reduction in serum sulfite levels after HD treatment.This suggests that at
least a part of serum sulfite exists asa dialyzable form. Although neutrophil
activation commonly occursduring HD, from our current available data the rate
of sulfiteproduction during dialysis is unknown, because we have not measured
sulfitecontained in the dialysate.
Although sulfite is widely used as preservative and antioxidantin food,
beverages, and pharmaceuticals, excessive sulfite ishighly toxic for human
cells and tissues. An extreme exampleis a congenital disease of sulfite
oxidase deficiency
(4,13,14).
Individualssuffering from this genetic disorder develop severe neurologic
abnormalities,dislocated ocular lenses, mental retardation, attenuated growth
ofthe brain, and early death
(4,13,14).
It is not clear whetherthe brain damage occurs as a result of toxic levels of
sulfite,the absence of sulfate, or a combination of both. A recent studyby
Reist et al. (8)
showed that sulfite exerts toxic effectson cultured neuronal cells directly
or in combination with peroxynitrite.Another target organ of sulfite is the
lung. It has been wellestablished that exposure to sulfite can cause
bronchial asthmaand other chronic lung diseases
(6). Its damaging effects to
thelung have been proposed to involve the generation of sulfiteradicals such
as SO3.-, SO4.-, and
SO5.-, as well as inactivationof
1-antiproteinase
(7,9).
Sulfite was also demonstrated todirectly activate neutrophils, leading to
enhanced migrationand generation of oxygen radicals
(25,26,27).
Thus, the sulfiteconcentration must be tightly regulated to maintain
homeostasisin humans. It remains unknown whether a rise (up to 10-fold
increaseabove normal range) in serum sulfite plays a role in organ andtissue
dysfunction in CRF patients. Oral sulfite loading inhealthy subjects results
in a transient increase in serum sulfiteconcentration to 38 to 112 µM in
30 min that returnedto basal levels within 3 h without any adverse reactions
(18).However, the possibility
cannot be ruled out that chronic andsustained elevation of serum sulfite in
CRF patients may bemore harmful than acute and transient rise in healthy
individuals.More information regarding the effects of sulfite on cellular
functionand precise determination of sulfite levels in tissues and organs
willbe necessary to address this issue.
Acknowledgments
We thank Dr. David Rothstein (Yale University, New Haven, CT)for critical
review of this manuscript.
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Received for publication April 9, 1999.
Accepted for publication August 11, 1999.
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[Abstract][Full Text][PDF]
Top
Abstract
Introduction
1-antiproteinase
(7,9).
Sulfite was also demonstrated to directly activate neutrophils, leading to
enhanced migration and generation of oxygen radicals
(25,26,27).
Thus, the sulfite concentration must be tightly regulated to maintain
homeostasis in humans. It remains unknown whether a rise (up to 10-fold
increase above normal range) in serum sulfite plays a role in organ and tissue
dysfunction in CRF patients. Oral sulfite loading in healthy subjects results
in a transient increase in serum sulfite concentration to 38 to 112 µM in
30 min that returned to basal levels within 3 h without any adverse reactions
(18). However, the possibility
cannot be ruled out that chronic and sustained elevation of serum sulfite in
CRF patients may be more harmful than acute and transient rise in healthy
individuals. More information regarding the effects of sulfite on cellular
function and precise determination of sulfite levels in tissues and organs
will be necessary to address this issue. 
