| Title | Double-Blind Study of Bio-normalizer Effects on Oxygen Radical Production and Carbohydrate Binding with Circulating Blood Leukocytes in Patients with Insulin-Independent Diabetes Mellitus |
|---|---|
| Year | |
| Author | Michail Balabolkin, Ludmila Korkina, Ashot Mkrtumyan |
| Publisher |
Final Report
Clinical Trial
Double-Blind Study of Bio-normalizer Effects on Oxygen Radical Production and Carbohydrate Binding with Circulating Blood Leukocytes in Patients with Insulin-Independent Diabetes Mellitus
Research supervisors
Professor Michail Balabolkin, Ph.D., Dr.Med.Sci., Director,
Institute of Diabetes, Russian Academy of Medical Science,
Moscow, Russia
Professor Ludmila Korkina, M.D., Ph.D., Dr.Med.Sci
Russian Institute of Pediatric Hematology, Moscow, Russia
Clinical Investigator
Dr. Ashot Mkrtumyan, M.D., Dr.Med.Sci.
Introduction
Earlier: we have performed pilot clinical trial, in which the clinical efficacy, free radical and immune-modulating activity of Bio-normalizer (BN) in the treatment of insulindependent diabetes mellitus patients have been studied. It was found that BN administration improved significantly clinical conditions in more than 80% patients, the markers of diabetes mellitus being decreased in patients’ blood and urine. The daily requirement of insulin decreased in average by 20-25%. We have shown for the first time that insulin injections suppressed the generation of reactive oxygen species by circulating leukoytes that may be regarded as a causative reason for a high risk of bacterial and fungal infections in diabetic patients. Along with beneficial clinical effects, BN therapy enhanced significantly (up to the normal value) the level of superoxide production by circulating monocytes and neutrophils as well as activity of nitric oxide synthase. On the grounds of these results, we concluded that surprisingly enough BN may act not merely as an antioxidant but appear to be a stimulator of enzymatic systems producing the physiologically essential oxygen radicals such as nitric oxide and superoxide. NO is synthesized in the reaction of L-arginine oxidation by a calcium activated NADPH-dependent NO-oxidase, which is blocked by L-arginine analogues N-monomethyl-L-arginine and N-iminoelhyl-L-ornithine [1]. This enzyme was found in endolhelial and neuronal cells, and in macrophages, neutrophils and monocyles after their “priming” with bacterial or yeast polysaccharides [1, 2]. It has been reported also that nitric oxide and superoxide radicals are produced simultaneously by cells isolated from an inflammatory exudate [3]. Those two free radicals interact with a rate constant of 6.7×109 M-1 s-1 to form peroxynitrite anion, which is known as a strong oxidant with bactericidal activity [4]. This decomposes spontaneously producing hydroxyl-like radicals, which could initiate lipid peroxidation [5]. Several studies have suggested that elevated glucose levels in both diabetic patients and experimental animals can generate oxygen radicals and cause membrane and lipoprotein lipid peroxidation [6, 7, 8]. Although the role of NO in inflammation is not quite clear yet, it was suggested that it can modulate the edema formation, increase chemotaxis of cells, and inactivate an excess; of superoxide derived from inflamed neutrophils [3]. Interestingly enough, that inhibition of NO-synthase by N-monomethyl-L-arginine leads to abolishion of the normally potent bactericidal activity of macrophages [9].
NO causes relaxation of smooth muscle by activation of soluble guanylate cyclase that leads in turn to increased cyclic 3′,5′-guanosine monophosphate (GMP) level [10].
The idea that oxidative stress contributes to diabetic complications such as vascular and renal dysfunctions is rather attractive because imbalance of constrictor (superoxide) and relaxing (NO) factors could result from variations in oxidant status. Josefsen et al., [11] have shown that the circulating monocytes in newly diagnosed type I diabetes patients are activated in terms of chemotaxis and superoxide production. At the same time they failed to find any changes in the monocyte functions 6 months after the primary diagnosis of IDDM. Hiramatsu and Arimori [12] have clearly shown that significantly elevated rate of superoxide generation in the diabetic hypertriglyceridemic monocytes was a reflection of a hypertriglyceridemic condition and did not correlate with the diabetic symptoms. If hyperglycemia itself is the cause of the diabetic complications, then it seems to be reasonable to suggest that elevated glucose level is toxic and that increased plasma and tissue glucose is a main source of the oxidative stress apparent in diabetes. Thus, glucose-dependent oxidative stress was found to be critical for determining cell damage in cultures [13]. Besides, oxygen free radicals enhance release of vasoconstrictor prostanoids or/and act as a direct vasoconsrictor factor. They can also inactivate NO, leading to excessive vasoconstriction. However, the exact role and mechanism of an excessive oxygen radical production in diabetes is yet to be determined. A clearer understanding of these processes may provide a more rational approach to the pharmacological combating of the diabetes-caused complications. Diabetes mellitus is known to be associated with extensive disease of both large and small blood vessels [14]. Because of that, diabetic patients often develop cardiomyopathy characterized mainly by left ventricular contractile dysfunction and congestive heart failure [15]. Diabetic microangiopathy includes diabetic retinopathy, diabetic glomerulosclerosis, and diffuse cappilary disease. Actually, no real progress has been made in prevention or treatment of progressive angiopathy in diabetes. Vascular disease accounts for the majority of the clinical complications of diabetes mellitus. Changes in local control of vascular tone such as unbalanced production of relaxing and contracting factors by the endothelial or/and the blood circulating cells may lead to the initiation and maintenance of abnormal vascular reactivity which is characteristic feature in diabetic patients. The endothelial and circulating cells participate in the local regulation of vascular smooth muscle tone by generating vascular relaxing factors such as nitric oxide (NO) and leukotriene E4, D4, and others, as well as vascular contracting factors including thromboxane A2 and superoxide anion [16].
To our knowledge little is known about redox state of diabetic patients treated by various sugar-decreasing drugs. A few last studies [17, 18] have shown that free radical-damaging processes are probably of essential importance for this type of diabetes due to the overproduction of oxygen radicals by monocytes and granulocytes. So far, we became curious to find out the in vivo features of oxidative stress such as oxygen radical overproduction by monocytes, which might be induced by elevated level of glucose itself but insulin injections. A main goal of the present clinical laboratory study was to obtain further evidence, if any, on the BN regulatory activity regarding oxygen radical production by circulating monocytes of patients with insulin-independent diabetes mellitus of both types.
The success of our previous trial made it possible to organize a new pilot study of BN administration for the insulin-independent diabetes mellitus patients. Since the effects of BN on clinical efficacy and immuno-modulating activity of diabetes patients have already been studied thoroughly in the first trial, we paid special attention to its effects on superoxide and nitric oxide formation and the expression and affinity of glucose- and galactose-binding receptors on the surface of circulating monocytes. Nonetheless, in accord with our previous observations, we registered a significant improvement of clinical conditions of most patients participated in the trial.
Patients and Study Design
Randomized double-blind study was carried out at Institute of Diabetes of Russian Academy of Medical Science. 24 adult patients suffered from insulin-independent diabetes mellitus (IIDM, Type I and type II) were randomized into two groups. Experimental group contained 15 patients and control group – 9 patients. 9 adult healthy people were in the reference group. The experimental group patients were treated with BN (2 sachets a day, daily) for 3 weeks. The control group patients were given 2 sachets containing plain sugar powder. The following parameters were measured in the beginning and after cessation of clinical trial luminol-dependent chemiluminescence of blood monocytes, NO-synthase activity and NO production by blood monocytes, number and affinity of the glucose and galactose receptors on the monocyte membrane. Eligibility criteria for the patients participating in the trial were similar to those in the previous trial with insulin-dependent diabetes patients (See Final Report on “The study of Bio-Normalizer (BN) clinical efficacy and immunomodulating activity in the treatment of insulin-dependent diabetes mellitus”).
Materials and Methods
Practically all the reagents including chemiluminescence probes luminol and lucigenin, activators of the cell oxygen radical-generating enzymes 12-O-phorbol-13-myristate acetate (PMA) and zymosan, inhibitor of NO-synthase N-monomethyl-L-arginine, enzymes CuZnSOD, :MnSOD, and catalase, adrenaline, Monoprep, Ficoll-Hepaque, tris-HCl buffer, polyacrylamide gel (P AAG), and Hank’s balanced salt solution were purchased from Sigma Co, St. Louis. Monoclonal antibodies to the lymphocyte and monocyte subpopulations were from Serva, Germany. Glucose- or galactose-containing PAAG conjugates were synthesized and kindly gifted by Prof. Bovin (Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia).
Blood drawing. Venous blood (10 ml) was obtained by venipuncture using heparinized vacutainers (Becton-Dickenson, Rutherford, USA) in the morning before any food. Freshly drawn blood was used for the further cell isolation.
White blood cells (WBC) and mononuclear cell isolation. White blood cells (WBC) were isolated from 10 ml blood sample, which was layered onto an equal volume of the mixture containing dextran sulfate (6% in potassium phosphate buffer) and metrizoate (32%) in ratio 5:2 v/v. To sediment erythrocytes, samples were incubated at 37° C for 30 min. Then the WBC-rich supernatant was placed on the top of Monoprep® solution and centrifuged at 150 x g for 40 min, and a thin layer between Monoprep solution and plasma, containing mononuclear leukocytes (monocytes and lymphocytes) was carefully collected with a Pasteur pippette and washed twice in a cold Hank’s balanced salt solution (HBSS). Finally, mononuclear cells were resuspended in a minimal essential medium (MEM) supplemented with 5% of fetal calf serum [19].
Production of reactive oxygen species (ROS) by monocytes. The release of ROS from monocytes was measured by the chemiluminescence (CL) method19 on a LKB 1251 chemiluminometer (Sweden) at 37° C under continuous mixing. A mononuclear cell suspension (105 cells) was added into a polystyrene cuvette containing 1 ml preheated HBSS with 50µM luminol (LDCL) and incubated for 5-6 min. The intensity of spontaneous CL was recorded continuously. After that, 1 mg/ml of zymosan particles opsonized by human serum proteins were added to cells, and the amplitude of the CL response to the activator was measured as a difference between the maximal intensities 6 of the activated and spontaneous CL. The results were expressed as mV per 106 monocytes [19].
Activity of NO-synthase was determined using L-arginine monomethyl ester (L NAME) as a specific inhibitor of the enzyme activity. Briefly, LDCL of monocytes was measured in the absence and in the presence of 50 mM of L-NAME. The inhibition of LDCL expressed in percent (%) corresponds to the nitric oxide synthase activity.
NO production. Nitric oxide generation by monocytes was measured spectrophotometrically using reaction of the phenol nitration. Monocytes (2×106 cells/ml) were suspended in the 50mM potassium phosphate buffer in the presence of phenol red (5 mM) and FeEDTA (2.5 mM) and stimulated with PMA (100 ng/ml). After incubation at 37°C for 2 hours, reaction was stopped by centrifugation at 300 x g for 10 min. Supernatant was collected, and its pH was adjusted to pH 6.2-6.5 by 1M HCl, and then to pH 10.0-10.6 by 3M NaOH. Absorbance at 420 nm was measured at each pH value. The amount of NO2-phenol formed was calculated using the pH-dependent difference in absorbance’s (∆Ɛ = 18300 M-1 cm-1).
A number and affinity of carbohydrate receptors were determined using polyacrylamide-glucose (PAAG-glu) and polyacrylamide-galactose (PAAG-gal) conjugates loaded with fluorescein. PAAG conjugates were obtained as described in [20] and contained the polyacrylamide molecules with the polymerization equal 100. The amide groups were partly substituted by glycosyle-OCH2CH2CH2, where glycosyl was the residue of glucose or galactose. The content of carbohydrates in the PAAG conjugates was equal 30 mol. %. The expression of carbohydrate receptors on the surface of mononuclear leukocytes was determined by flow cytometry using a FACScan (Becton Dickinson, USA). Isolated cells were fixed in the 1% paraformaldehyde solution and then used for the flow cytometry measurements. Five thousand cells from each sample were scanned and the results were analyzed automatically with a PC and the Facscan Software. To discriminate a non-specific spontaneous fluorescence in cells, the internal control was performed during each measurement. The control cell samples were processed as the experimental ones hut in the absence of the fluorescein-loaded conjugates. The mean values of the fluorescence intensity and the number of fluorescent cells were determined. The results were expressed in the arbitrary units and per cent, respectively [20].
Statistical analysis
Dynamics of laboratory parameters before and after the BN administration in both experimental and control groups were analyzed using of the non-parametric Wilcockson criteria. The results for the experimental and control groups were expressed as mean ± S.E.M. Statistically significant difference was assumed at the 5% level.
Results and Discussion
Type II diabetes, noninsulin dependent diabetes mellitus, or maturity-onset diabetes, is on the rise, affecting more than 100 million people worldwide. In The United States alone disease incidence has more than doubled in the past 20 years, and 500,000 new cases are expected every year. India reports 30 million patients [21]. Currently, the term “type II diabetes” (IIDM) means that there is no indications of autoimmune disease. While, “type I diabetes, indicates the presence of autoimmune markers such as islet cell or glutamic acid decarboxylase antibodies. Meantime, type II diabetes is defined as a syndrome capable of being initiated by a number of causes. A condition known as “insulin resistance” is thought to be the initiating disorder. Normally, pancreatic beta cells secrete insulin enough to maintain circulating blood glucose levels. On one hand, insulin inhibits glucose production in liver and on the other hand, it stimulates glucose binding to circulating leukocytes, which deliver glucose to sceletal muscles. In insulin resistance, the muscles become unavailable to uptake glucose, some is reversed into fat, and the rest continues to circulate in the blood. The circulating blood glucose levels reach those of type I diabetes. At this point, the body faces a physiological paradox: both insulin and glucose levels are high and patients have already exhibited the micro and macro vascular disorders, which cause severe complications such as heart attacks, angina, strokes, and amputations. The researchers and physicians have been searching for years now for new preparations relevant to diminish the “insulin resistance” syndrome and ameliorate to certain extent the vascular complications in both type I and type II diabetes mellitus.
What makes cells insensitive to insulin remains unclear. However, we suggested that impaired balance of oxygen radicals produced by circulating leukocytes may easily cause oxidation of proteins on the cellular surface that tums out to change the sugar binding receptor pattern. In this study we showed that monocytes of IIDM patients produced an abnormal amount of reactive oxygen species that was revealed by the chemiluminescence approach. Indeed, the monocytes of some patients a two-fold increased level of spontaneous LDCL as compare to normal one (Table 1). Moreover, the intensity of zymosan-activated LDCL in diabetic patients was about 5 times greater than that of healthy donors. A short term BN administration lowered both spontaneous and activated LDCL practically to the normal levels (Table 1). At the same time, there were patients with dramatically decreased levels of spontaneous and activated LDCL. After taking BN, the monocytes of those patients produced practically normal amount of oxygen free radicals (Table 2). (In monocytes of the placebo group patients the initially increased spontaneous LDCL decreased, though rather high zymosan activated LDCL further increased after the clinical trial cessation, Table 3). The double effect of BN could be explained in terms of (1) its well-established free radical scavenging activity and (2) its potency to stimulate cellular enzymes either producing or utilizing the oxygen free radicals. Thus, it has been shown in a number of studies that lipopolysaccharides of bacterial and yeast origin possessed the ”priming effect” towards NADPH-oxidase as well as they can induce manganese SOD and NO-synthase, via the production of interleukin-1 and TNF-α. [22,23,24]. BN, enriched with the yeast polysaccharides may be regarded as an inducer of some initially depressed enzymatic systems. However, we found that NO production by circulating monocytes was significantly higher in the IIDM patients comparing with healthy donors (Table 4). In this case, BN administration suppressed NO generation by cells. In the placebo group there were not any changes in the levels of NO produced by cells upon stimulation (Table 5). As nitric oxide is able to interact with superoxide radical forming peroxinitrate, which is thought to be the most powerful endogenous toxicant [3], it could be assumed that beneficial effects of BN on the IIDM patients is a consequence of its superoxide- and nitric oxide-scavenging activity.
Completely new data were obtained studying the binding of PAAG conjugates with the monocyte membrane. It was found that a number of cells bound to the PAAG-glucose and PAAG-galactose conjugates was lower for diabetic patients in comparison with donors (Tables 6 and 8). The course of BN therapy improved situation for both saccharides (glucose and galactose). Unfortunately, we failed to find any abnormalities in binding of both conjugates with monocytes of the placebo group patients in the beginning of trial (Tables 7 and 9). Therefore, it is very hard to make a definite conclusion on the difference found in the experimental and control groups. We could only assume that BN is capable increasing the impaired carbohydrate binding to monocyte membrane that allows to diminish glucose levels in blood plasma.
Conclusion
1. BN administration improved significantly patients’ clinical conditions and their quality of life. This conclusion is based on the patients’ and physicians’ observations.
2. BN administration resulted in normalization of oxygen radical production by blood monocytes: it inhibited initially enhanced oxygen radical generation in patients with IIDM of both types. At the same time, BN administration induced an elevation of initially suppressed oxygen radical generation by monocytes from patients with IIDM. Thus finding can be considered as an universal molecular mechanism by which BN improves the clinical conditions in patients with diabetes mellitus.
3. BN administration inhibited the increased nitric oxide production by blood monocytes in IIDM patients. As a consequence, BN administration normalized the vessel contraction/relaxation and permeability.
4. BN administration increased the carbohydrate binding with monocytes that in turn improves the glucose utilization and its transportation to tissues.
5. These results provide a theoretical basis for BN implementation in diabetic patients with vascular and infectious complications.
6. As we know from the previous studies that BN supplementation significantly lowers blood lipid peroxidation products, it could be beneficial in reducing risk of cardiovascular disease in diabetic patients.
References
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2. Ischiropoulos H., Zhu L., and Beckman J.S. Peroxynitrite formation from macrophage-derived nitric oxide. Arch. Biochem. Biophys., 1992, 298, 446-451
3. Rodenas J., Mitjavila M.T., and Carbonell T. Simultaneous generation of nitric oxide and superoxide by inflammatory cells in rats. Free Rad. Biol. 11ed1.995, 18, 5, 869- 875
4. Zhu L., Gunn C., and Beckman J.S. Bactericidal activity of peroxynitrite. Arch. Biochem. Biophys., 1992, 298, 452-457
5. Radi R., Beckman J.S., Bush K.M., and Freeman B.A. Peroxynitrite-induced membrane lipid peroxidation in human low density lipoprotein. Free Rad.Res. Comms., 1992, 17, 9-20
6. Jain S.K. and Levine S.N. Elevated lipid peroxidation and vitamin E-quinone levels in heart ventricles of streptozotocin-treated diabetic rats. Free Rad. Biol. Med., 1995, 18, 2, 337-341
7.Rajeswari P., Natarajan R., Nadler J.L., Kumar D., and Kaltra V.K. Glucose induces lipid peroxidation and inactivation of membrane-associated ion-transport enzymes in human erythrocytes in vivo and in vitro. J. Cell Physiol., 1991, 149, 100-109
8.Oberley L.W. Free radicals and diabetes. Free Rad. Biol. Med., 1988, 5, 113-124.
9. Yaqoob M., Patrick A.W., McClelland P., Stevenson A., Mason H.,White M.C., and Bell G..M. Relationship between markers of endothelial dysfunction, oxidant injury and tubular damage in patients with insulin-dependent diabetes mellitus. Clin.Sci.Colch., 1993, 85, 5, 557-562.
10. McCall T.B., Freelisch M., Palmer R.M., and Moncada S. Identification of N iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. Br. J. Phatmacol., 1991, 102, 234-238.
11. Josefsen K., Nielsen H., Lorentzen S., Damsbo P., and Buschard K. Circulating monocytes are activated in newly diagnosed type I diabetes mellitus patients. Clin. Exp. Immunol., 1994, 98, 3, 489-493
12. Hiramatsu K., and Arimori Sh. Increased superoxide production by mononuclear cells of patients with hypertriglyceridemia and diabetes. Diabetes, 1988, 37, 832-837
13. Donnini D., Zambito A.M., Pen-ella G., Ambesi-lmpiombato F.S., and Curcio F. Glucose may induce cell death through a free radical-mediated mechanism. Biochem. Biophys. Res. Communs. 1996; 219(2): 412-417.
14. Bloodworth, J.M.B., Jr. Diabetic microangiopathy. Diabetes 12: 99-114; 1963
15. Fein, F.S.; Sonnenblick, E.H. Diabetic cardiomyopathy. Proc. Cardiovas. Dis. 27: 255-270; 1985
16. Tesfamariam B. Free radicals in diabetic endothelial cell dysfunction. Free Rad. Bioi. Med., 1994, 16, 3, 383-391
17. Delamaire Ivl., Maugendre D., Moreno M., Le Goff M.C., Allanic H., and Genetet B. Exploration des differentes etapes du fonctionnement des polynucleares neutrophiles chez les patients diabetiques. J. Mal. Vase. 1995; 20: 107-120.
18. Griesmacher A., Kindhauser M., Andert S.E., et al., Enhanced serum levels of thiobarbituric-acid-reactive substances in diabetes mellitus. Am. J. Med. 1995; 98: 469-
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19. Korkina, L.G.; Samochatova, E.V.; Maschan, A.A.; Suslova, T.B.; Cheremisina, Z.P.; Manas’ev, l.B. Release of active o:-s:ygen radicals by leukocytes of Fanconi anemia patients. J.Leukocyte Bioi. 52: 357-362; 1992.
20. Mikhalchik E.V., Korkina L.G., Shiyan S.D., and Bovin N.V. Effect of neoglycoconjugates on the in vitro luminol-dependent chemiluminescence of rat peritoneal macrophages. Membr. and Cell Biol. 1995; 8: 607-614.
21. Edington S.M. Amiloid plaque and diabetes. Bio/Technology 1994; 12: 591-594.
22. Visner G.A., Dougall W.C., Wilson J.M., Burr I.A., and Nicks H.S. Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumour necrosis factor. Role in the acute inflanunatory response. J.Biol.Chem. 1990; 265:2856-2864.
23. Nakayama D.K., Geller D.A., Lowenstein C.J., Davies P., Pitt B.R., Sinunons R.L., and Billiar T.R. Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat puhnonary artety smooth muscle. Am.J.Respir. CellJvfol.Biol. 1992; 7: 471-476.
24. Geller D.A., Nussler A.K., Silvio M.D., Lowenstein C.J., Shapiro R.A., Wang S.C., Sinunons R.L., and Billiar T.R. C)1okines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc.Natl.Acad.Sci. USA 1993; 90: 522-526.
Table 1. Effect of BN on the intensity of spontaneous and zymosan-activated luminol dependent chemiluminescence (LDCL) by blood monocytes (Experimental subgroup of patients with enhanced LDCL level)
| Patient | Type of diabetes |
Intensity of spontaneous LDCL (mV/106 cells) |
Intensity of zymosan- activated LDCL (mV/106cells) |
||
| before | after | before | after | ||
| Ka-ova | II | 1160 | 188 | 16910 | 1630 |
| Va-tzov | II | 750 | 95 | 13560 | 162 |
| Mel-ko | II | 1030 | 201 | 23670 | 1919 |
| Ko-in | I | 1060 | 1540 | 15320 | 35660 |
| So-ka | II | 670 | 760 | 10470 | 2900 |
| Tem-ov | I | 790 | 630 | 17120 | 3740 |
| Rav-ich | II | 860 | 1160 | 15590 | 6260 |
| Af-in | II | 1400 | 440 | 23200 | 11000 |
| Ef-ov | I | 1440 | 520 | 54300 | 960 |
|
m±SD |
1018 ± 222 | 615 ± 362 | 21127 ±8398 | 7137 ± 1969 | |
|
Normal-values |
525 ± 215 |
4216 ± 2461 |
|||
Table 2. Effect of BN on the intensity of spontaneous and zymosan-activated LDCL by blood monocytes. (Experimental subgroup with diminished LDCL level)
| Patient | Type of diabetes |
Intensity of spontaneous LDCL (mV/106 cells) |
Intensity of zymosan- activated LDCL (mV/106cells) |
||
| before | after | before | after | ||
| Kr-ova | I | 178 | 220 | 3855 | 4161 |
| Ta-an | II | 220 | 1100 | 3300 | 8780 |
| Sa-av | II | 30 | 360 | 810 | 1420 |
| Ko-yaev | II | 88 | 360 | 810 | 1420 |
| Kar-eva* | II | 40 | 700 | 1180 | 4540 |
| Obr-ova | II | 30 | 420 | 394 | 2400 |
|
m±SD |
98 ± 68 | 527 ± 249 | 1725 ± 1235 | 3787 ± 2040 | |
|
Normal-values |
525 ± 215 |
4216 ± 2461 |
|||
* – Insulin-dependent patient
Table 3. Effect of placebo on LDCL on blood monocytes.
|
Patient |
Type of |
Spontaneous |
Zymosan-activated LDCL |
||
| before | after | before | after | ||
| Pro-na | I | 900 | 510 | 8500 | 26990 |
| Ibra-ova | II | 760 | 320 | 7300 | 8100 |
| Ali-va | II | 1190 | n.a | 3070 | n.a |
| Sil-aya | II | 1020 | 780 | 7810 | 2920 |
| Vet-aya | II | 2620 | 380 | 34680 | 1480 |
| Shu-ov | II | 360 | 1000 | 3040 | 4400 |
| Per-chay | II | 1150 | 360 | 3750 | 760 |
| Alyu-va | II | 1540 | 1200 | 7590 | 4050 |
|
m±SD |
1192.5 ± 452 | 650 ± 487.9 | 8045 ± 643.4 | 15520 ± 16221 | |
|
Normal-values |
525 ± 215 |
4216 ± 2461 |
|||
Table 4. Effect of BN administration on nitric oxide production by blood monocytes.
(Expetimental group)
|
Patient
|
Type of
|
NO-synthase inhibition (%) |
NO-production |
||
| before | after | before | after | ||
| Kru-ovana | I | 60.4 | 88.2 | n.a | n.a |
| Kas-va | II | 38.9 | 44.2 | n.a | n.a |
| Vas-tzov | II | 41.3 | 40.1 | n.a | n.a |
| Mel-ko | II | 36.2 | 48.2 | n.a | n.a |
| Kos-in | I | 43.5 | 40.6 | 45.1 | 8.6 |
| Sor-ka | II | 54.8 | 43.4 | 43.9 | 6.6 |
| Tem-ov | I | 47.1 | 58.6 | 29.5 | 53.3 |
| Rav-vich | II | 36.4 | 33.9 | 27.0 | 34.6 |
| Ef-ov | I | 48.2 | 69.8 | 15.0 | 17.3 |
| Ta-an | II | 44.8 | 46.9 | 28.0 | 13.6 |
| Sav-va | II | 39.3 | 47.9 | 28.9 | 20.3 |
| Kon-ev | II | 43.5 | 45.8 | 36.7 | 8.2 |
| Kar-eva | II* | 44.9 | 69.7 | 27.3 | 7.7 |
| Af-in | II | 43.8 | 51.6 | 15.4 | 9.7 |
| Obr-ova | II | 39.6 | 52.5 | 28.9 | 18.8 |
|
m±SD |
43.0 ± 1.3 | 50.7 ± 2.7 | 29.6 ± 6.7 | 18 ± 10 | |
|
Normal-values |
51.5 ± 0.6 |
14.1 ± 4.4 |
|||
Table 5. Nitric oxide production in the control (placebo) group
|
Patient
|
Type ofdiabetes
|
NO-synthase inhibition (%) |
NO-production |
||
| before | after | before | after | ||
| Kru-ovana | I | 60.4 | 88.2 | n.a | n.a |
| Kas-va | II | 38.9 | 44.2 | n.a | n.a |
| Vas-tzov | II | 41.3 | 40.1 | n.a | n.a |
| Mel-ko | II | 36.2 | 48.2 | n.a | n.a |
| Kos-in | I | 43.5 | 40.6 | 45.1 | 8.6 |
| Sor-ka | II | 54.8 | 43.4 | 43.9 | 6.6 |
| Tem-ov | I | 47.1 | 58.6 | 29.5 | 53.3 |
| Rav-vich | II | 36.4 | 33.9 | 27.0 | 34.6 |
| Ef-ov | I | 48.2 | 69.8 | 15.0 | 17.3 |
| Ta-an | II | 44.8 | 46.9 | 28.0 | 13.6 |
| Sav-va | II | 39.3 | 47.9 | 28.9 | 20.3 |
| Kon-ev | II | 43.5 | 45.8 | 36.7 | 8.2 |
| Kar-eva | II* | 44.9 | 69.7 | 27.3 | 7.7 |
| Af-in | II | 43.8 | 51.6 | 15.4 | 9.7 |
| Obr-ova | II | 39.6 | 52.5 | 28.9 | 18.8 |
|
m±SD |
43.0 ± 1.3 | 50.7 ± 2.7 | 29.6 ± 6.7 | 18 ± 10 | |
|
Normal-values |
51.5 ± 0.6 |
14.1 ± 4.4 |
|||
Table 6 Effect of BN administration on biding of the polyacrylamide-glusoce conjugates with monocyte membrane. (Experimental group)
|
Patient
|
Type of
|
Intensity of Fluorescence |
Number of florescent cells (%) |
|||
| before | after | before | After | |||
| Kos-in | I | n.a | 337 | 3.5 | 10.4 | |
| Sor-ka | II | n.a | 371 | 2.9 | 2.1 | |
| Tem-ov | I | n.a | 363 | 2.5 | 2.5 | |
| Rav-vich | II | n.a | 442 | 2.0 | 2.2 | |
| Ef-ov | I | 312 | 374 | 3.9 | 3.4 | |
| Ta-an | II | 315 | 384 | 4.4 | 3.3 | |
| Sav-va | II | n.a | n.a | n.a | n.a | |
| Kon-ev | II | 36.1 | 374 | 1.3 | 5.1 | |
| Kar-eva | II* | 351 | 363 | 1.2 | 4.3 | |
| Af-in | II | 321 | n.a | 4.8 | 7.0 | |
| Obr-ova | II | 367 | 347 | 1.1 | 5.7 | |
|
m±SD |
343± 7 | 373 ± 10 | 2.8 ± 1.1 | 4.6 ± 1.9 | ||
|
Normal-values |
341±30 |
3.6±1.1 |
||||
Table 7 binding of fluorescein-labeled polyacrylamide-glucose conjugates to the monocyte membranes (placebo group).
|
Patient
|
Type of |
Intensity of Fluorescence |
Number of florescent-labeled cells (%) |
||
| before | after | before | After | ||
| Pro-na | I | 391 | 396 | 4.6 | 2.4 |
| Ibra-va | II | 369 | 315 | 6.8 | 3.1 |
| Al-va | II | 323 | n.a | 1.0 | n.a |
| Sil-aya | II | 352 | n.a | 1.6 | n.a |
| Vetr-aya | II | 329 | 386 | 5.3 | 2.2 |
| Shu-ov | II | 336 | 374 | 3.0 | 0.7 |
| Per-chay | II | 306 | 263 | 1.1 | 3.3 |
| Sel-va | II | 338 | 349 | 2.9 | 1.4 |
| Alush-va | II | 308 | 274 | 2.6 | 4.7 |
|
m±SD |
343± 23 | 337 ± 45 | 3.2 ± 1.6 | 2.5 ± 1.0 | |
|
Normal-values |
341±30 |
3.6±1.1 |
|||
Table 8 Effect of BN administration on binding of fluorescein-labeled polyacrylamide-galactose conjugates to the monocyte membranes (experimental group).
|
Patient
|
Type of |
Intensity of Fluorescence |
Number of florescent-labeled cells (%) |
|||
| before | after | before | After | |||
| Kos-in | I | 363 | 492 | 10.2 | 14.7 | |
| Sor-ka | II | 413 | 427 | 7.2 | 7.4 | |
| Tem-ov | I | 385 | 489 | 6.8 | 10.8 | |
| Rav-vich | II | 412 | 460 | 5.6 | 9.6 | |
| Ef-ov | I | 367 | 370 | 5.2 | 15.5 | |
| Ta-an | II | 417 | 476 | 4.3 | 15.8 | |
| Sav-va | II | 405 | 443 | 3.6 | 6.7 | |
| Kon-ev | II | 471 | 555 | 3.9 | 14.3 | |
| Kar-eva | II* | 409 | 471 | 4.2 | 15.0 | |
| Af-in | II | 453 | 494 | 5.2 | 12.1 | |
| Obr-ova | II | 406 | 485 | 3.1 | 12.4 | |
|
m±SD |
409.2 ±21.8 | 469.3 ± 32.2 | 5.4 ± 1.5 | 12.2 ± 2.6 | ||
|
Normal-values |
478±27 |
10.0±3.2 |
||||
409.2±2
Table 9. Binding of fluorescein-labeled polyacrylamide-galactose conjugates to the monocytes membrane (the placebo group).
|
Patient
|
Type of |
Intensity of Fluorescence |
Number of florescent-labeled cells (%) |
||
| before | after | before | After | ||
| Pro-na | I | 440 | 393 | 13.5 | 9.7 |
| Ibra-va | II | 490 | 433 | 18.5 | 12.2 |
| Al-va | II | 479 | n.a | 8.6 | n.a |
| Sil-aya | II | 368 | 502 | 11.1 | 19.7 |
| Vetr-aya | II | 495 | 466 | 18.8 | 6.1 |
| Shu-ov | II | 422 | 451 | 9.8 | 8.0 |
| Per-chay | II | 495 | 406 | 6.1 | 11.0 |
| Sel-va | II | 385 | 448 | 9.4 | 9.4 |
| Alush-va | II | 361 | 425 | 9.9 | 14.0 |
|
m±SD |
437± 47 | 440 ± 26 | 11.7 ± 3.4 | 11.3 ± 3.0 | |
|
Normal-values |
478±27 |
10.0±3.2 |
|||