JURNAL SUBDIVISI PEDIATRY GAWAT DARURATJURNAL SUBDIVISI PEDIATRY GAWAT DARURAT

SIZE AND CHARGE SIZE AND CHARGE CHARACTERISTICS OF THE CHARACTERISTICS OF THE PROTEIN LEAK IN DENGUE PROTEIN LEAK IN DENGUE SHOCK SYNDROMESHOCK SYNDROME

Bridget. A. Wills, Emmannuelle E. Oragui, Nguyen Minh Dung. Ha Thi Loan, Nguyen Vinh Bridget. A. Wills, Emmannuelle E. Oragui, Nguyen Minh Dung. Ha Thi Loan, Nguyen Vinh Chau, Jeremy J. Ferrar and Michael Levin.Chau, Jeremy J. Ferrar and Michael Levin.

Lenny Syntia DewiLenny Syntia Dewi

BackgroundBackground. The mechanism underlying the . The mechanism underlying the transient vascular leak syndrome of dengue transient vascular leak syndrome of dengue hemorrhagic fever (DHF) is unknown. We aimed to hemorrhagic fever (DHF) is unknown. We aimed to determine whether molecular size and charge determine whether molecular size and charge selectivity, which help restrict plasma proteins selectivity, which help restrict plasma proteins within the intravascular space, are altered in within the intravascular space, are altered in patients with DHF and whether a disturbance of patients with DHF and whether a disturbance of the anonic glycosaminoglycan (GAG) layer on the the anonic glycosaminoglycan (GAG) layer on the luminal endothelial surface constributes to disease luminal endothelial surface constributes to disease pathogenesis.pathogenesis.

Methods.Methods. We measured serial plasma We measured serial plasma levels and fractional clearences of proteins levels and fractional clearences of proteins with different size and charge with different size and charge characteristics in 48 children with dengue characteristics in 48 children with dengue shock syndrome (DSS) and urinary shock syndrome (DSS) and urinary excretion profiles of heparan sulfate, excretion profiles of heparan sulfate, chondroitin-4-sulfate, and chondroitin-6-chondroitin-4-sulfate, and chondroitin-6-sulfate in affected children and healthy sulfate in affected children and healthy control subjects.control subjects.

Results.Results. Compared with convalesent values, Compared with convalesent values, acute plasma concentration of all proteins were acute plasma concentration of all proteins were reduced, with increased fractional clearances. reduced, with increased fractional clearances. Smaller proteins were more affected than larger Smaller proteins were more affected than larger molecules. Albumin, which is normally protected molecules. Albumin, which is normally protected from leakage by its strong negative charge, from leakage by its strong negative charge, demonstrated a clearance pattern similar to that demonstrated a clearance pattern similar to that of transferrin, a neutral molecule of similar size. of transferrin, a neutral molecule of similar size. Urinary heparan sulfate excretion was Urinary heparan sulfate excretion was significantly increased in children with DSS.significantly increased in children with DSS.

Conclusion.Conclusion. The endothelial size- The endothelial size-dependent sieving mechanism for plasma dependent sieving mechanism for plasma proteins is at least partially retained, proteins is at least partially retained, whereas selective restriction base on whereas selective restriction base on negatice charge is impaired. The increase negatice charge is impaired. The increase heparan sulfate excretion suggests a role heparan sulfate excretion suggests a role for GAGs in the pathogenesis of the for GAGs in the pathogenesis of the vascular leak.vascular leak.

INTRODUCTIONINTRODUCTION 20 th century : emergence of 20 th century : emergence of

dengue as one of the principal dengue as one of the principal infectious disease of the infectious disease of the trofical world trofical world

WHO : 2,5 billion people live WHO : 2,5 billion people live in areas of risk, between 250 in areas of risk, between 250 and 500,000 cases of Dengue and 500,000 cases of Dengue Hemorrhagic Fever (DHF) Hemorrhagic Fever (DHF)

1998 epidemic in Southern 1998 epidemic in Southern Vietnam alone almost 120,000 Vietnam alone almost 120,000 cases of DHF and 342 deaths cases of DHF and 342 deaths most of which occurred in most of which occurred in childrenchildren

The pathological mechanism underlying The pathological mechanism underlying the capillary leak are poorly understood. the capillary leak are poorly understood.

Many factors : including virus burden, viral Many factors : including virus burden, viral virulence, host immune response, and virulence, host immune response, and genetic predisposition-have ben implicated genetic predisposition-have ben implicated in the pathogenesis of DHF, but the in the pathogenesis of DHF, but the means by which any of these factors might means by which any of these factors might influence endothelial function remain influence endothelial function remain unclear. unclear.

Glycosaminoglycans (GAGs) are complex, Glycosaminoglycans (GAGs) are complex, negatively charged polisaccharides that are negatively charged polisaccharides that are widely distributed on cell surfaces and are widely distributed on cell surfaces and are incorporated into the glycocalyx layer on the incorporated into the glycocalyx layer on the luminal surface of the vascular luminal surface of the vascular endothelium. endothelium.

Disruption of the GAG components of the Disruption of the GAG components of the glycocalyx layer has been implicated in the glycocalyx layer has been implicated in the increased clearance of proteins seen in increased clearance of proteins seen in animal models of capillary leakanimal models of capillary leak

The pathogenesis of renal protein-losing The pathogenesis of renal protein-losing disorders, such as steroid responsive-disorders, such as steroid responsive-nephrotic syndrome (SRNS) and nephrotic syndrome (SRNS) and generalized vascular leak syndrome such generalized vascular leak syndrome such as meningococcal septicemiaas meningococcal septicemia

No reseach has been done to investigate to No reseach has been done to investigate to investigate whether distruption of the GAG investigate whether distruption of the GAG component of the glycocalyx layer might be component of the glycocalyx layer might be feature of the capillary leak syndrome in feature of the capillary leak syndrome in DHF. DHF.

This study to measure the urinary This study to measure the urinary excreation profiles of the 3 major GAGs excreation profiles of the 3 major GAGs (heparan sulfate, chondroitin-6-sulfate and (heparan sulfate, chondroitin-6-sulfate and chondroitin-4-sulfate) that are important in chondroitin-4-sulfate) that are important in maintaining the permselective properties maintaining the permselective properties of the capillary wall in the same group of of the capillary wall in the same group of children and compared the results with children and compared the results with those from healthy control subjects.those from healthy control subjects.

Fractional urinary clearances for the Fractional urinary clearances for the various proteins were calculated relative to various proteins were calculated relative to that of creatinine as follows :that of creatinine as follows :

Fractional clearance of protein.Fractional clearance of protein.

= [(U= [(UprotprotV/PV/Pprotprot)/(U)/(UcreatcreatV/PV/Pcreatcreat)] x 100)] x 100

PATIENTS, MATERIALS AND METHODSPATIENTS, MATERIALS AND METHODS

48 children DSS since 1998-1999 48 children DSS since 1998-1999 (table 1)(table 1)PICU Hospital for Tropical Diseases of Ho Chi Minh City, VietnamPICU Hospital for Tropical Diseases of Ho Chi Minh City, Vietnam

Ringer LactatRinger Lactat 1 or 2 colloids1 or 2 colloids

Simultaneous plasmaSimultaneous plasma urin samplesurin samples

Before resusitation the following day at a 1 monthBefore resusitation the following day at a 1 month(day 1) (day 2) follow up visit(day 1) (day 2) follow up visit

Storage -70oC Storage -70oC storage – 30oCstorage – 30oC

Protein studies, Protein studies, GAG studies, GAG studies, acted as their own controlsacted as their own controls healthy school provided control urine sampleshealthy school provided control urine samples

Laboratory MethodLaboratory Method - transported on dry ice to St.Mary’s Hospital London- transported on dry ice to St.Mary’s Hospital London -Inactivated with the virucidal agent Triton X-100-Inactivated with the virucidal agent Triton X-100

Statistical AnalysisStatistical Analysis

Table 1. World Health Organization guidelines for the Table 1. World Health Organization guidelines for the clinical diagnosis of clinical diagnosis of dengue shock syndrome in endemic dengue shock syndrome in endemic

areas areas

CriteriaCriteria NotesNotes

FeverFever 2 – 7 days2 – 7 days

Hemorrhagic TendencyHemorrhagic Tendency Any of the following positive tourniquet test result, Any of the following positive tourniquet test result, spontaneous petechiae or other skin bleeding or mucosal or GIT tract bleedingspontaneous petechiae or other skin bleeding or mucosal or GIT tract bleeding

ThrombocytopeniaThrombocytopenia Platelet count Platelet count ≤ 100,000/mm≤ 100,000/mm3 *Evidence of plasma leakage Any of the following increase in admisson hematokrit to

≥ 20 %, above the expected mean for age, sex and population, decrease in hematocrit to ≥20 % of the baseline value after resuscitation or clinical signs of plasma leakage, such as pleuran effusion or ascites

Circulatory compromise narrow pulse pressure ≤ 20 mmHg, with tachycardia or hypotension for age

* A few patients, although thrombocytopenic did not have absolute platelet counts ≤ 100.000/mm3 at the time of admission

RESULTSRESULTS

Table 2. Admission characteristics of 48 children with Table 2. Admission characteristics of 48 children with dengue shock syndrome, presented according to severitydengue shock syndrome, presented according to severity

of shock as assessed atof shock as assessed atdischarge or deathdischarge or death

Level of shockLevel of shock

MildMild Moderate/severeModerate/severe

CharacteristicCharacteristic (n =28) (n =28) (n = 20) (n = 20) p* p*

Age,median (90 % range)years 9 (4- 14)Age,median (90 % range)years 9 (4- 14) 8 (3 – 13) NS 8 (3 – 13) NS

Male sex, no (%)Male sex, no (%) 12 (43) 9 (45) 12 (43) 9 (45) NS NSDay of illness when presenting with shock, median (90 % range) 5 (3 -8)Day of illness when presenting with shock, median (90 % range) 5 (3 -8) 5 (3 – 10) 5 (3 – 10) NS NS

Unrecordable pulse, no (%)Unrecordable pulse, no (%) 2 (7) 2 (10) NS (F) 2 (7) 2 (10) NS (F)

Pulse rate, median (90 % range), beats/min**Pulse rate, median (90 % range), beats/min** 110 (88 – 130) 120 (110 – 140) 110 (88 – 130) 120 (110 – 140) 0,01 0,01

Unrecordable blood presure, median (90 % range)mmHg** 1 (4) 4 (20) NS (F)Unrecordable blood presure, median (90 % range)mmHg** 1 (4) 4 (20) NS (F)

Systolic blood pressure, median (90 % range) mmHg** 100 (80-120)Systolic blood pressure, median (90 % range) mmHg** 100 (80-120) 90 (80-100) 90 (80-100) 0,1 0,1

Admission pulse pressure, no (%)Admission pulse pressure, no (%)

≤ ≤ 10 mmHg10 mmHg 10 (36) 10 (36) 6 (30) NS 6 (30) NS

> 10 ≤ 20 mmHg> 10 ≤ 20 mmHg 18 (64) 18 (64) 14 (70) 14 (70) NS NS

Spontaneous bleeding, no (%)Spontaneous bleeding, no (%) 24 (86) 15 (75) NS (F) 24 (86) 15 (75) NS (F)

Hematocrit, median (90 % range) %Hematocrit, median (90 % range) % 49 (42 – 57) 50 (40-57) NS 49 (42 – 57) 50 (40-57) NS

Platelet count, median, (90 % range), cells.mm3 98.000 70.000 NSPlatelet count, median, (90 % range), cells.mm3 98.000 70.000 NS

(15.000-150.000) (9000-180.000) NS(15.000-150.000) (9000-180.000) NS

Note , NS not significantNote , NS not significant• For categorical variables, X2 test or Fisher exact test (F), For continuous variable, nonparametric Mann Withney U testFor categorical variables, X2 test or Fisher exact test (F), For continuous variable, nonparametric Mann Withney U test

** For those with recordable values** For those with recordable values

Figure 1 Figure 1

The levels of hypoproteinemia were The levels of hypoproteinemia were profound, and the fractional protein profound, and the fractional protein clearancesclearances

Figure 1. Serial plasma levels and fractional urinary clearances for IgG, transferrin, and albumin in children with acute dengue shock syndrome, by day of shock, where day 1 is the day of presentation with shock. Comparisons for days 1 and 2 blood ( $n=38$ and 39) and urine ( $n=27$ and 34) results for the combined severity groups with convalescent values were all highly significant ( $P< .001$ , Wilcoxon signed-rank test for paired data). Within-day comparisons between the mild shock and moderate/severe shock groups were significant for all 3 plasma protein concentrations ( $P< .01$ , Mann-Whitney U test) but not for the urinary clearances. Dotted arrows, approximate ranges seen in acute meningococcal sepsis [25].

Figure 2. Left, fractional urinary clearances of antithrombin, by day of shock. Days 1 and 2 clearances were significantly higher than convalescent values ( $P< .001$ , Wilcoxon signed-rank test for paired data), but there were no differences between shock severity groups. Right, correlation between antithrombin and albumin clearances for the day-2 results (Spearman correlation, .8; $P< .001$ ).

Data for the urinary excreation of the 3 Data for the urinary excreation of the 3 GAGs are presented in GAGs are presented in table 3table 3

TABLE 3. Urinary glycosaminoglycan : creatinine TABLE 3. Urinary glycosaminoglycan : creatinine excreation ratios for children with acute Dengue Shock excreation ratios for children with acute Dengue Shock

Syndrome (DSS) vs. healthy control subjectsSyndrome (DSS) vs. healthy control subjects

Heparan sulfate,Heparan sulfate, Chondroitin-4-sulfateChondroitin-4-sulfate Chondroitin-6-Sulfate Chondroitin-6-Sulfate

GroupGroup mg/mmol creatininemg/mmol creatinine mg/mmol creatininemg/mmol creatinine mg/mmol creatinine mg/mmol creatinine

Acute DSSAcute DSS 0,47 * (0,19 – 0,78) 0,37 (0,12 – 0,67)0,47 * (0,19 – 0,78) 0,37 (0,12 – 0,67) 0,54 (0,21 – 0,78) 0,54 (0,21 – 0,78)

Healthy Control Subjects Healthy Control Subjects 0,23 (0,06 – 0,52) 0,31 (0,12 – 0,46)0,23 (0,06 – 0,52) 0,31 (0,12 – 0,46) 0,48 (0,31 – 2,02) 0,48 (0,31 – 2,02)

NOTE. Data are median (80 % range)NOTE. Data are median (80 % range)

* P = .016 vs. control group (Mann-Whitney U Test)* P = .016 vs. control group (Mann-Whitney U Test)

DISCUSSIONDISCUSSION

The consentrations of a series of proteins The consentrations of a series of proteins of different molecular size are all markedly of different molecular size are all markedly reduced in the plasma of children reduced in the plasma of children presenting with acute DSS and that this presenting with acute DSS and that this reduction correlates with clinical severity.reduction correlates with clinical severity.

There is a corresponding increase in There is a corresponding increase in fractional urinary clearances of the same fractional urinary clearances of the same proteins and have confirmed that leakage proteins and have confirmed that leakage contributes significally to the reduction in contributes significally to the reduction in plasma concentration in plasma plasma concentration in plasma concentration of the anticoagulant protein concentration of the anticoagulant protein antithrombin.antithrombin.

The smaller proteins (MW 59,000-79,000 The smaller proteins (MW 59,000-79,000 Da) were more affected than IgG (MW Da) were more affected than IgG (MW 150,000 Da) 150,000 Da)

Stronge correlations between proteins of Stronge correlations between proteins of similar size similar size ~ usual size-dependent ~ usual size-dependent sieving mechanism is at least partially sieving mechanism is at least partially retained. retained.

Clearances of albumin, which is usually Clearances of albumin, which is usually protected from leakage by its stronge protected from leakage by its stronge negative charge, and transferrin, a neutral negative charge, and transferrin, a neutral molecule, correlated closely with each molecule, correlated closely with each other, which suggests that the selective other, which suggests that the selective restriction based on negative charge may restriction based on negative charge may be impaired. be impaired. ~ endothelial glycocalyx DSS~ endothelial glycocalyx DSS

CONCLUSIONCONCLUSION

At present, volume overload, which may be At present, volume overload, which may be exacerbated by the leakage of resuscitation exacerbated by the leakage of resuscitation fluids, constitutes one of the major complications fluids, constitutes one of the major complications of DSS that contributes to mortality.of DSS that contributes to mortality.

Finally, if the molecular mechanisms responsible Finally, if the molecular mechanisms responsible for the increased vascular permeability were for the increased vascular permeability were better understood, it might be possible to design better understood, it might be possible to design pharmacological intervensions to counteract or pharmacological intervensions to counteract or prevent leakageprevent leakage

TerimakasihTerimakasih

GLYCOCALYXGLYCOCALYX

Wessler and whiteman method :Wessler and whiteman method :

- GAG- GAG

- Hyaluronic acid- Hyaluronic acid

- Chondroitin- Chondroitin

- - Chondroitin-4-sulphateChondroitin-4-sulphate

- Chondroitin-6-sulphate- Chondroitin-6-sulphate

- Dermatan sulphate- Dermatan sulphate

- Heparin- Heparin

- - Heparan SulphateHeparan Sulphate

- Heparan Sulphate I, II- Heparan Sulphate I, II

- Proteoglycan - Proteoglycan

Crockroft dan Gault kreatinin clearance Crockroft dan Gault kreatinin clearance orang yang tingginya lebih dari 5 kaki, bila orang yang tingginya lebih dari 5 kaki, bila pengumpulan urin sukar atau dalam pengumpulan urin sukar atau dalam keadaan mendesak :keadaan mendesak :

pria : Kr Cl = pria : Kr Cl = 140 – umur x BBI140 – umur x BBI ml/menit ml/menit

72 x B72 x B

wanita : Kr Cl = Kr Cl x 0,85 ml/menitwanita : Kr Cl = Kr Cl x 0,85 ml/menit

BBI = Berat Badan IdealBBI = Berat Badan Ideal

B = kadar kreatinin darah dalam mg/dlB = kadar kreatinin darah dalam mg/dl

Bromocresol green methodBromocresol green method

Principle Principle At pH=4.2, albumin bind At pH=4.2, albumin bind

with with bromocresolbromocresol greengreen to produce a to produce a

blue-blue-greengreen complex. The complex. The change in absorbance at change in absorbance at 628 nm628 nm

correlates with the correlates with the concentration of albumin. concentration of albumin.

Alkaline Picrate MethodAlkaline Picrate Method

Beckman CoulterBeckman Coulter

The recognition that urinary leak can The recognition that urinary leak can provide useful information about systemic provide useful information about systemic capillary function, together with the ability capillary function, together with the ability of reliable techniques to document small of reliable techniques to document small changes in protein clearance, present a changes in protein clearance, present a novel and interesting approach to try to novel and interesting approach to try to unravel the complexities of dengue unravel the complexities of dengue pathophysiology and thereby improve pathophysiology and thereby improve case management. case management.