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 Moscow State Medical Stomatological University, Moscow. 199034 Russia

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

AbstractThe 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 Clinical Hospital with hydronephrosis resulting from vesicoureteral reflux or urinary obstruction. The children and their relatives had no pathology linked with hemorrhagic diathesis or thrombosis. Operations were performed with intubation narcosis and were equivalent in surgical intervention and duration. The children were divided into three groups according to their clinical condition and the results of routine laboratory tests. Group 1 included 12 children hospitalized in a relatively satisfactory condition: the body temperature and the results of laboratory tests (complete blood count, urine analysis, and biochemical blood analysis) were within physiological ranges. Pyelonephritis associated with hydronephrosis was in remission. The postoperative period proceeded without complications.

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 38C; 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 (Israel) and the computer program Gold-Rada, version 3.0. The radiopharmaceutical tracer (RT) Tc-99m was injected intravenously at a dose of 80 MBq. Dynamic scintigraphy makes it possible to assess the separate and total excretory functions of the kidneys, urodynamics, and the anatomical features and topography of the kidneys.

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 IIVI) 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 IIIII. 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.

 


REFERENCES

1.     Brande, H., Opal, S.M., Vogel, S.N., and Morrison, D.C., Endotoxin in Health and Disease, New York, Basel, 1999.

2.     Yakovlev, M.Yu., Elements of Endotoxin Theory of Human Physiology and Pathology, Fisiol. Chel., 1999, vol. 29, no. 4, p. 98.

3.     Yakovlev, M.Yu., Endotoxin Aggression As a Predisease or a Universal Factor of Pathogenesis of Diseases in Humans and Animals, Usp. Sovrem. Biol., 2003, vol. 123, no. 1, p. 31.

4.     Rabi, K., Lokalisovannaya i rasseyanaya vnutrisosudistaya koagulyatsiya (Local and Disseminated Intravascular Coagulation), Moscow, Meditsina, 1974.

5.     Permyakov, N.K., Yakovlev, M.Yu. and Shlyapnikov, V.V., Acute Renal Insufficiency (Role of Endotoxin in Pathogenesis), Pat. Fisiol. 1989, no. 6, p. 77.

6.     Westphal, O., Luderitz, O., Galanos, C et al., The Story of Endotoxin, Proc. 2nd Int. Conf. Adv. lmmunopharm., 1975, p. 13.

7.     Zenkevich, O.D., Anikhovskaya, I.A., Yakovlev. M.Yu., et al., RF Patent 2093825. 2002.

8.     Urazaev, R.A., Yakovlev, M.Yu., Anikhovskaya. I.A.. et al., RF Patent 2011993, 1994.

9.     Likhoded, V.G., Yakovlev, M.Yu., Apollonin. A.V., et al., RF Patent 2088936, 1997.

10. Koblov, L.F., Metody i pribory dlya issledovaniya gemostaza (Assays and Instruments for Hemostasis). Moscow, Meditsina, 1975.


 

Table 3. Disturbances of hemostasis as dependent on the condition of the endotoxin-antiendotoxin system

 


Variant

Plasma LPS,

EU/ml

Anti-ET antibodies, AU

Reserve of LPS binding by granulocytes

%

Am, arb. units

T1, s

T2, s

T, s

Ao, arb. units

T3, s

FA, %

to Re glycolipid

to general antigen

I

(n = 20)

0.19 0.03

200 10

400 20

4.9 0.2

3.0 0.1

211 6.9

343 15

551 14

0.4 0.03

0

23 2.1

 

 

 

 

Normal coagulation

II

(n = 34)

1.6 0 1*,**

214 23

302 24#

1.0 0.3*

3.5 0.1*

212 19

185 31*

397 46#, **

0*

603 109*,**

40 4.5 **

 

 

 

 

Compensated hypercoagulation

III

(n = 29)

2.1 0.15*

218 31

305 32

0.6 0.3*

3.3 0.2

215 24

143 26*,**

358 48* **

0*

∞*,**

0* **

 

 

 

 

Noncompensated hypercoagulation

IV

(n = 4l)

2.2 0.25*

226 26

342 35

0.6 0.2*

3.3 0.2

258 21

546 51*,**

803 55*

0.10.02*,**

295 38*,**

22 5.3**

 

 

 

 

Subcompensated hypercoagulation

V

(n = 43)

3.4 0.3*,**

307 27*,**

398 38##

0.75 0.3*,**

3.4 0.1*

284 25*

528 45*

788 41*

0.8 0.1*,**

40 12*,**

44 3.0*,**

 

 

 

 

Noncompensated hypocoagulation with macrohematuria in 30 % of cases

VI

(n = ll)

5.8 0.4*,**

130 28**

258 29*,**

0*,**

3.0 0.1

205 44

517 7.0*

738 58*

0.6 0.1

30 15*

53 6.0*

 

 

 

 

Noncompensated hypocoagulation with macrohematuria in 100% of cases

 

Note: The difference from the normal value was significant at (*) P < 0.001 or (#) P < 0.05. The difference from the other variant was significant at (**) P < 0.001 or (##) P < 0.05