Biochemical changes in polymorphonuclear leucocytes in diabetic patients.JM Sawant
Dept of Biochemistry, Seth GS Medical College, Parel, Bombay, Maharashtra.
A study on the functional ability of polymorphonuclear leucocytes (PMNL) indicates that the total lysosomal enzyme levels viz. Beta-glucuronidase, lysozyme, acid phosphatase and alkaline phosphatase were not altered in diabetics, compared to that in control subjects. However, the findings also reveal that the release of these lysosomal enzymes in response to a particulate stimulus is impaired in diabetics. This suggests that the bactericidal capacity of these cells, which are involved in phagocytosis, is impaired in diabetics, making them more vulnerable to infections.
Keywords: Adult, Case-Control Studies, Diabetes Mellitus, complications,Female, Human, Infection, etiology,Lysosomes, chemistry,Male, Neutrophils, chemistry,Phagocyte Bactericidal Dysfunction, blood,epidemiology,etiology,Recurrence, Support, Non-U.S. Gov′t,
Diabetic patients are more prone to infections and show high morbidity and mortality rates. Prior to the advent of modern antimicrobial therapy, infection accounted for much of the morbidity in diabetics. A correlation between susceptibility to infection and control of diabetes is noted in other studies, suggesting that the poor diabetic control, particularly in presence of ketoacidosis, is associated with lowered resistance to infection and may be due, in part, to impaired leucocyte function.
A number of studies,,, have now been carried out to identify the defect in host defense mechanism in diabetic patients. The primary abnormalities appear to be in polyrn orphon u clear leucocyte function and are more prominent in poorly controlled diabetics. Three different aspects of polymorphonuclear leucocytes (PMNL) function have been examined in these studies viz. chemotaxis, phagocytosis and bactericidal activity. The bactericidal activity was studied by microbiological methods against Staphylococcus aureus and Escherichia More Details Coli and selected strains of Streptococci.
In the present work carried out at University hospital a new biochemical technique was used for estimating the release of lysosomal enzymes in response to particulate stimuli, in order to assess the bactericidal capacity of these cells, which may also focus light on the defect in the mechanism involved in the bactericidal activity.
The study group comprised of poorly controlled diabetic patients, of both sexes, between 20-40 years of age, attending King Edward Memorial Hospital, Mumbai. The diabetics were detected by their fasting blood sugar and post glucose blood sugar levels. Healthy persons of both sexes, in the same age group formed the controls for the study. Heparinized venous blood samples were collected from all the subjects.
1. Isolation of PMNL: PMNL were isolated by the method of Aguado and Pujol and were suspended in phosphate buffered saline (PBS) supplemented with CaCI2 and MgCI2.
2. Cell viability was assessed by trypan blue dye exclusion method at three different stages viz. (i) after isolation of PIVINL, (ii) after treatment with cytochalasin B and (iii) after treatment with serum treated zymosan (STZ).
3. STZ was prepared as described previously , and used as particulate stimulus for PMNL.
4. The cell suspension was divided into four aliquots viz.
1st aliquot : Cells treated with cytochalasin B (Sigma Chemical Co.) and STZ;
2nd aliquot : cells treated with only STZ;
3rd aliquot : cells treated with triton X-100 for lysis
4th aliquot : untreated cells.
Each aliquot was treated as described previously.
5. Measurement of lysosomal enzyme release: After treatment with STZ, the reaction mixtures were centrifuged and cell free supernatants were separated for enzyme assays. Beta-glucuronidase was estimated by the method of Brittinger et al using phenolphthalein glucuronic acid as a substrate. Lysozyme was estimated by measuring the rate of lysis of Micrococcus lysodeikticus (Sigma Chemical Co.) in terms of change in absorbance at 450 nm. Acid phosphatase was estimated by the technique of Kind and King. Alkaline phosphatase was measured by the method of King et al.
6. The cytoplasmic enzyme, Lactate dehydrogenase (LDH), was measured by the kinetic method of Wacker et al
Statistical analysis: The results were expressed as Mean ? SD. The comparison of diabetic group with control group was done by Student's T test.
There were 20 poorly controlled diabetic patients of both sexes in the study group and 23 healthy persons in the control group.
The cell viability was found between 95-97%. The cell viability observed after treatment with cytochalasin B and STZ was between 95-96%. This is also supported by the activities of LDH as shown in [Table - 1], which shows no change in LDH activity after treatment with cytochalasin B and/or STZ compared to the untreated cells. The total LDH activity was similar in both diabetic and control subjects.
Exposure of PMNL to STZ caused release of lysosomal enzymes in both diabetic, as well as, control subjects. This release of lysosomal enzymes was prominent in cytochalasin B pretreated cells [Table - 2].
The total enzyme activities of beta glucuronidase, alkaline phosphatase and acid phosphatase were not changed in diabetics compared to controls; however, the activity of lysozyme was decreased, to some extent, in diabetics (p < 0.05).
The lysosomal enzyme release in response to STZ by cells pre-treated and untreated with cytochalasin B was reduced in diabetics compared to controls as evident from the enzyme activities of aH lysosomal enzymes studied which showed highly significant reduction (p < 0.001 [Table - 2]).
The percent release of all lysosomal enzymes by cells of diabetic and control subjects, untreated and untreated with cytochalasin B, in response to STZ is shown in [Table - 2]. An overall decrease in release of lysosomal enzymes was observed in diabetic patients when compared to controls.
In a previous study, the phenomenon of lysosomal enzymes release in response to particulate stimuli in control subjects has been reported. These enzymes possess bactericidal activity.
In PMNI- isolated from diabetic patients, the total lysosomal enzyme activities were not significantly altered. The selective release of lysosomal enzymes on exposure to STZ and not of cytoplasmic enzymes was noted in both diabetic as well as control groups. However, significant reduction in release of lysosomal enzymes was observed in diabetic patients compared to control subjects. This release of lysosomal enzymes in response to STZ is due to the phenomenon known as "regurgitation during feeding",. The PMNL pre-treated with cytochalasin B, a fungal metabolite, evoked enhanced response to STZ attributed to the process of "reverse endocytosis"- in diabetic as well as control subjects. A significant reduction in enzyme release occurred in diabetic subjects compared to controls.
In diabetics, there was a reduction in the release of lysosomal enzymes, on exposure of PMNL to particulate stimuli, even in the presence of normal total lysosomal enzymes activities." This may indicate that the functional capacity of these cells to destroy micro-organism by the process of phagocytosis 'is decreased.
The Phagocytic function of PMNL in diabetic individuals has been evaluated in a number of studies. Bybee and Rogerss demonstrated significant suppression of phagocytic activity in patients with diabetic ketoacidosis, but not in non-ketotic diabetics. A similar defect in phagocytosis was found by Bagdade and coworkers in the PMNL of poorly controlled diabetics. In their subsequent study, they reported the phagocytic and bactericidial ability of the PMNL for pneumococci. A defect was noted in both these functions, which was reversible with insulin treatment. The authors believe that insulin is necessary for the normal metabolism ofglucose to provide energy for the process of phagocytosis and destruction of microorganisms.
The process of phagocytosis, which is an energy consuming process, is fuelled by ATP derived from glycolytic and Hexose Monophosphate shunt pathways. The derangement in the carbohydrate metabolism in diabetic may be the reason for the impairment in the bactericidal ability of PMN leucocytes of poorly controlled diabetic patients making them more susceptible to recurrent infections.
The author is thankful to Dr (Mrs) SP Taskar, ex-Professor in the Dept. of Biochemistry, Seth GS Medical College for her guidance in this study and to the Dean, Seth GS Medical College and King Edward Memorial Hospital for providing necessary facilities in the CVTC Laboratory of the Institute. Financial assistance was received from CSIR, New Delhi in the form of Jr. research fellowship to the author to carry out the work.
[Table - 1], [Table - 2]