Intestinal
Endotoxin in Regulation of Hemostasis Activity
and in Pathogenesis of the DIC Syndrome
M. V. Meshkov1,2,
I.A. Anikhovskaya3, M.M. Yakovleva3, and M.Yu. Yakovlev3
1
2 lzmailovo Children's Clinical Hospital, Moscow, Russia
3 Institute of General and Clinical Pathology, Clinical Diagnostic
Society,
Russian Academy of Natural Sciences, Moscow, 127093 Russia
Received
December 24, 2004
Abstract—The role of the kidneys in endotoxin aggression (EA) and the involvement
of EA in disturbance of hemostasis were shown in 33 children with congenital
urological pathology. With a progressive decrease in the accumulative-excretory
function of the kidneys, compensated chronic EA became subcompensated
or non-compensated with a pronounced clinical manifestation: fever,
exacerbation of chronic pyelonephritis, an expanded
form of the disseminated intravascular coagulation (DIC) III syndrome, and macrohematuria. Preoperative correction of antiendotoxin immunity and hemostasis and improvement of
urine passage prevented bleeding and other postoperative complications.
Endotoxin (ET) of
gram-negative bacteria is a lipopolysaccharide (LPS)
with a broad spectrum of useful and pathogenic properties [1]. The biological
action of ET depends on the ET concentration in the blood and the activity of antiendotoxin immunity (AEI) [2]. An excessive inflow of
interstitial LPS into the blood in absolute or relative deficiency of AEI
results in endotoxin aggression (EA), which causes various syndromes and
diseases [3].
In the middle of the
last century, Rabi [4] postulated the disseminated intravascular coagulation
(DIC) syndrome for a rabbit model of Shwartzman's
phenomenon (a double intravenous injection of ET with a 24-h interval) in
which the kidneys are the most affected organs, causing acute renal
insufficiency and death of experimental animals [5]. This fact is fundamentally
important because the kidneys have the leading role in excretion of LPS from
the blood during experimental EA [6]. On this basis, we thought it interesting
to study the dynamics of parameters of ET in the blood serum, activity of AEI,
and hemostasis in subjects with different degrees of disturbance of the
accumulative-excretory function of the kidneys. We believe that such data may
provide useful information about the participation of LPS in the activity of
hemostasis and in pathogenesis of its disturbances as well as about the role of
the kidneys in excretion of ET from the blood and the body.
METHODS
The study involved 33
children aged from 3 months to 14 years who were hospitalized in the lzmailovo Children's
In groups 2 and 3, hydronephrosis was associated with the acute phase of
chronic pyelonephritis. The subjects of group 2 (14
children) were hospitalized in a moderately grave condition with mild pyrexia
without leukocytosis but with a significantly
decreased amount of band neutrophils and an increased
erythrocyte sedimentation rate (ESR). Urinalyses revealed 18-20 leukocytes
per visual field. Blood urea and creatinine were
within physiological ranges. All subjects received antibacterial and infusion
therapy during the pre- and post- operative periods. In all children of group
2, the early postoperative period was complicated with the hemorrhagic
syndrome in the form of macrohematuria for 3-8 days,
sometimes associated with blood clots in the urinary bladder and in drainages
and with bleeding from the area of postoperative sutures. The operative area
was revised in two children with persistent hemorrhage. A bleeding vessel was
not found, and the whole surface of the operative wound was bleeding diffusely.
Hemostatic therapy under the control of a dynamic electrocoagulogram was successful in all cases.
All seven children of
group 3 were hospitalized in grave condition: the body temperature was higher
than 38°C; the amount of leukocytes was within physiological ranges, but
juvenile forms were absent; lymphocytosis and eosinophilia were revealed; and the ESR was sharply
increased. In urinalyses, leukocytes completely covered the visual field.
Infusion therapy was performed for detoxification and correction of the blood
volume and the acid-base balance. In all subjects, percutaneous
nephrostomy or ureterostomy
or ureteral stenting were
performed two months before the operation because of their grave condition. In
six children, the postoperative period proceeded without the hemorrhagic
syndrome. Only in one child, aged 1 year and 8 months, with a kidney
malformation complicated by pyonephrosis and leukocytosis (12.610), was the postoperative period
complicated by macrohematuria in spite of the
performed nephrostomy. We linked it with an
insufficient nephrostomy period (two weeks) and an
early operation.
Along with the
standard clinical and laboratory tests, the LPS concentration was assayed in
serum repeatedly (five to seven times) by the clinically adapted Limulus
amebocyte lysate test (LAL
test) [7] using E-toxate (Sigma). Parameters of humoral AEI were assayed using a screening diagnostic
system (SOIS-ELISA) [8]. The granulocytic pan of AEI was studied using LPS
ELISA, which assayed the ET binding by leukocytes [9].
Hemostasis was
assayed repeatedly (five to seven times) with an H-334 electrocoagulograph.
This assay is inexpensive and available at any time and makes it possible to
correct therapy immediately.
Diagram of a normal electrocoagulogram. See
Methods for abbreviations.
The following
parameters of the electrocoagulogram were measured (figure).
T1 was the time corresponding to the
first phase of coagulation, the thrombin formation time, which is determined by
the ratio of pro- and anticoagulants; T2
was the time of fibrin formation under the action of thrombin (T2 depends on the amounts of platelets
and fibrinogen); T was the time of
all phases of coagulation (chronometric coagulation); Am
was the maximum amplitude, characterizing the hematocrit; Ao was the minimum amplitude, which
characterized the density of the blood clot and its resistance to fibrinolysis, which depends on the amount and quality of
platelets and fibrinogen; and A1 was
the amplitude observed 10 min after the beginning of retraction and fibrinolysis. The amplitudes, which were presented in
relative units, corresponded to the electro- conductivity of the blood. The
time of blood clot retraction and the beginning of fibrinolysis
(T3) and the relative fibrinolytic activity (FA), which was expressed as FA =A1 × 100%/Am, were studied according to Koblov [10].
Blood samples were taken by the standard method from fasting subjects before
and after the operation, on days 3-5 after the operation, and before the
discharge from the hospital.
The
accumulative-excretory function of the kidneys was studied by dynamic γ scintigraphy
with an L-Sting γ camera (
Statistical analyses
were performed using the program Excel 5.0.
RESULTS AND DISCUSSION
Based on the proposed
participation of intestinal LPS in the regulation of hemostasis and in
pathogenesis of the DIC syndrome, we divided the subjects into three groups
according to the disturbance of the accumulative-excretory function of the
kidneys (Table 1). In rabbits, the kidneys play an important role in
eliminating ET from the blood and the body and may determine the ET
concentration in the blood.
Table 1. Parameters of dynamic scintigraphy in
disturbances of the accumulative excretory function of the kidneys.
|
|
|
Degree of disturbance of renal functions |
||
|
|
Norm |
moderate |
pronounced
moderate |
severe |
|
|
|
(N=11) |
(N = 9) |
(N = 7) |
|
Tmax,
min |
5.2
± 0.4 |
9.3
±3.6 |
14.6
±2.4 |
19.5
± 2.2 |
|
|
|
P > 0.5 |
P < 0.05 |
P< 0.001 |
|
T 1/2, min |
12.0
±3.0 |
48.6
± 11.4 |
- |
- |
|
|
|
P< 0.001 |
P< 0.001 |
P< 0.001 |
|
Excretion
index, % |
51.0
±5.0 |
18.3
± 6.9 |
0 |
0 |
|
|
|
P< 0.001 |
P< 0.001 |
P<0.001 |
The data (Table 1)
demonstrated that, in the subjects with a moderate disturbance of the
accumulative- excretory function, a diminishing of excretion was more typical
than a diminishing of accumulation. The maximum RT accumulation time (Tmax) did not differ
significantly from the normal values (P > 0.5) and was 9.3 ± 3.6 min, while
the excretion half time (T1/2) was four times higher than normal (P
< 0.001). The excretion index was three times lower than normal (18.3 ±
6.9%, P < 0.001).
The functions of both
accumulation and excretion were decreased in pronounced moderate and severe
forms of renal disorders. Tmax for these forms amounted to 14.6 ± 2.4 and
19.5 ± 2.2 min, respectively, and significantly differed from normal values (P
< 0.05 and P < 0.001, respectively). T1/2
virtually did not dominate over the accumulation period. The excretion index
was 0%.
A comparative
analysis of the data of dynamic scintigraphy of the
kidneys, the LAL test, the clinical condition of
subjects, the body temperature, and the leukocyte count revealed a direct
relationship between the disturbance of the accumulative-excretory function and
the ET concentration in the blood (Table 2). The same relationship was found
for the condition of subjects and the body temperature.
Table 2. Relationship between the endotoxin activity, clinical condition,
body temperature, leukocyte count, and degree of disturbance of the
accumulative excretory function of the kidneys in subjects with urological
pathology.
|
Degree of disturbance |
Endotoxin activity, EU/ml |
Clinical condition |
Body temperature, °C |
Leukocyte count xlO
x 9/1 |
|
Moderate (N = 12) |
1.5
±0.2 |
Satisfactory |
36.6 ±0.25 |
7.0
± 0.4 |
|
Pronounced moderate (N = 12) |
2.0
± 0.2* |
Moderately grave |
37.5 ±0.2* |
7.0
±0.6 |
|
Severe (N = 9) |
5.3
± 1.2* |
Grave |
38.5 ±0.45* |
7.7
±0.8 |
Thus, the human kidneys
have the important function of ET excretion from the body. Increased
insufficiency of the excretory function (from the moderate to the severe form)
caused a progressive rise in the ET concentration in the blood (from 1.5 ± 0.2
to 5.3 ± 1.2 EU/ml, respectively, with significant differences between the
severe, pronounced moderate, and moderate forms, P < 0.05) and,
consequently, increased the body temperature since ET is an exclusive pyrogen [2, 3].
At first sight, the
leukocyte counts did not differ in different forms of kidney disorders and were
within physiological ranges. However, these values are decreased when
considered with respect to the subjects' condition, the body temperature, and
the ET concentration. The absence of juvenile leukocytes in groups 2 and 3
manifests an exhaustion of reserves of myelopoiesis
and suggests prolonged irritation of the bone marrow. The latter is probably a
direct result of chronic EA since ET is a multipotent
activator abolishing genome repression.
A comparative
analysis of all parameters before and after operation was performed to study
the possible relationship between the endotoxin-antiendotoxin
system and hemostasis. The results revealed a direct relationship between the
parameters (Table 3).
The rise in ET in the
blood (variants II—VI) was accompanied by an increase in the activity of the
coagulation-anticoagulation system, and the state of hemostasis changed from
compensated hypercoagulation (DIC I) to noncompensated hypocoagulation,
consumption coagulopathy (DIC III). In variant I,
the parameters were within physiological ranges. In variant II, an increase in
blood ET (1.6 ± 0.2 EU/ml) corresponded to a tendency towards an increased
concentration of antiglycolipid antibodies (214 ± 23
absorbance units, AU), a considerable decrease in antibodies to the general
antigen of enterobacteria (302 ± 24 AU), and
suppression of the granulocytic part of AEI (the reserves of ET binding
decreased to 1.0 ± 0.3%). The hemostasis system was characterized by
compensated chronometric and structural hypercoagulation
(decreased T = 397 ± 46 s, decreased Ao = 0 arb. units,
increased T3 = 603 ± 109 s, and
increased FA = 40.0 ± 4.5%). The changes observed in the second group cannot be
qualified as EA owing to the absence of their clinical manifestation
(hemostasis was compensated and the hemorrhagic syndrome was absent). Since an
eightfold increase in ET over the physiological value had no clinical
manifestation and was not accompanied by an increased activity of the humoral part of AEI, this condition was regarded as latent,
or compensated, chronic EA.
It is of interest to
study the dynamics of parameters of hemostasis with ET activity increasing to
about 2.0 EU/ml (variants III and IV). We revealed subcompensated
(T = 803 ± 55 s, Ao = 0.1 ± 0.02 arb.
units, T3 = 295 ± 38 s. and FA = 22 ±
5.3%) or noncompensated (T = 358 ± 48 s, Ao = 0 arb. units, T3 = ∞
and FA = 0%) chronometric and structural hypercoagulation
(DIC II) with no increase in the humoral part of AEI
and a diminished reserve of LPS binding by granulocytes. The condition of the
subjects with such parameters of ET, AEI, and hemostasis was qualified as subcompensated chronic EA, varying in severity. An ET blood
concentration close to 2.0 EU/ml was regarded as threatening DIC II.
Variants V and VI
were characterized by a progressive rise in blood LPS and development of noncompensated hypocoagulation,
which varied in severity and correlated directly with the blood ET
concentration and inversely with the activity of AEI. This condition was
denoted as noncompensated chronic EA, which, like subcompensated EA, could be divided into two forms. In
variant V, the LPS concentration amounted to 3.4 ± 0.3 EU/ml, the reserve of ET
binding by granulocytes was 0.75 ± 0.37c, and the titer of antiglycolipid
antibodies increased 1.5-fold (307 ± 27 AU). Chronometric and structural hypocoagulation (T
= 788 ±41 s, Ao - 0.8 ± 0.1 arb. units, T3 =
40 ± 12 s, and FA = 44 ± 3.0%) was accompanied in 30% of cases by short-term macrohematuria, which was readily stopped by volume and
electrolyte corrective agents, fresh frozen plasma, or aminocapronic
acid. In the second, more severe form of noncompensated
chronic EA, the average LPS concentration increased to 5.8 ± 0.4 EU/ml; the
titers of antiglycolipid antibodies, which neutralize
ET, decreased to 130 ± 28 AU; the reserve of ET binding by granulocytes fell to
zero; and consumption coagulopathy developed (T = 738 ± 58 s, Ao = 0.6 ± 0.1 arb.
units, T3 = 30 ± 15 s, and FA = 53 ± 6.0%)
and was always (in 100% of cases) associated with prolonged persistent macrohematuria (DIC III).
Thus, disturbance of
the accumulative-excretory function of the kidneys as a result of congenital
urological pathology is accompanied by chronic EA with exacerbations. The
moderate diminishing of the accumulative-excretory function causes an eightfold
rise in ET concentration in the blood (1.6 ± 0.2 EU/ml) with a tendency towards
a minor increase in the activity of the humoral part
of AEI, a significant decrease in the granulocytic part of AEI, and compensated
hypercoagulation. Yet this condition lacks clinical
manifestations and can be qualified as compensated chronic EA. A further
diminishing of the accumulative-excretory function (pronounced moderate and
severe forms) is accompanied by a progressive increase in ET concentration in
the blood and the appearance of a subcompensated or noncompensated form of EA with hyperactivation
of hemostasis and the DIC syndrome. AEI limits the
pathogenic effect of EA. Under conditions of only minimal reserves of AEI
(variant V), macrohematuria was not found in the
subjects, notwithstanding their grave condition, with hyperthermia and DIC
II—III. Only a sharp diminishing of the humoral part
of AEI and the absence of reserves of the granulocytic part of AEI result in
pronounced DIC III with macrohematuria. Thus, AEI,
along with ET, participates in regulation of the activity of hemostasis and in
pathogenesis of the DIC syndrome.
CONCLUSIONS
1)
Intestinal ET participates in regulation of the
activity of hemostasis and is an obligate factor in pathogenesis of the DIC
syndrome in children with congenital urological pathology associated with
renal accumulative-excretory insufficiency.
2)
The kidneys are important ET-eliminating organs whose
accumulative-excretory insufficiency causes EA in humans.
3)
The intensity of hemostatic
disorders and their response to medical correction depend directly on the ET
concentration in the blood and inversely on the activity of the humoral and granulocytic parts of AEI.
4)
Under conditions of a progressive diminution of the
accumulative-excretory renal function in children with congenital urological
pathology, compensated chronic EA becomes subcompensated
or noncompensated with a pronounced clinical
manifestation: fever, exacerbation of chronic pyelonephritis,
an expanded form of DIC III, and macrohematuria.
5)
Preoperative correction of AEI and hemostasis (fresh
frozen plasma, etc.) and the improvement of urine passage (nephrostomy
or stenting) prevents bleeding and other
postoperative complications and is worthy of improvement and development of the
respective algorithms.
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