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 ::  Abstract
 ::  Introduction
 ::  Methods
 ::  Results
 ::  Discussion
 ::  Acknowledgment
 ::  References
 ::  Article Tables

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Year : 1993  |  Volume : 39  |  Issue : 4  |  Page : 183-6

Biochemical changes in polymorphonuclear leucocytes in diabetic patients.

Dept of Biochemistry, Seth GS Medical College, Parel, Bombay, Maharashtra.

Correspondence Address:
J M Sawant
Dept of Biochemistry, Seth GS Medical College, Parel, Bombay, Maharashtra.

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Source of Support: None, Conflict of Interest: None

PMID: 0007996492

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 :: Abstract 

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,

How to cite this article:
Sawant J M. Biochemical changes in polymorphonuclear leucocytes in diabetic patients. J Postgrad Med 1993;39:183

How to cite this URL:
Sawant J M. Biochemical changes in polymorphonuclear leucocytes in diabetic patients. J Postgrad Med [serial online] 1993 [cited 2023 Mar 30];39:183. Available from:

  ::   Introduction Top

Diabetic patients are more prone to infections and show high morbidity and mortality rates[1]. 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[2],[3] 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[4],[5],[6],[7] 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 Scientific Name Search  and  Escherichia More Details Coli[3] and selected strains of Streptococci[8].

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.

  ::   Methods Top

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[9] and were suspended in phosphate buffered saline (PBS) supplemented with CaCI2 and MgCI2.

2. Cell viability was assessed by trypan blue dye exclusion method[10] 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 [11], 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[11].

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[12] 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[13]. Acid phosphatase was estimated by the technique of Kind and King[14]. Alkaline phosphatase was measured by the method of King et al[15].

6. The cytoplasmic enzyme, Lactate dehydrogenase (LDH), was measured by the kinetic method of Wacker et al[16]

Statistical analysis: The results were expressed as Mean ? SD. The comparison of diabetic group with control group was done by Student's T test.

  ::   Results Top

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.

  ::   Discussion Top

In a previous study[11], 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"[17],[13]. The PMNL pre-treated with cytochalasin B, a fungal metabolite, evoked enhanced response to STZ attributed to the process of "reverse endocytosis"[18]-[19] 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[6] in the PMNL of poorly controlled diabetics. In their subsequent study[7], 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[7].

The process of phagocytosis, which is an energy consuming process, is fuelled by ATP derived from glycolytic and Hexose Monophosphate shunt pathways[20]. 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.

  ::   Acknowledgment Top

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.

 :: References Top

1. Mufson MA, Kruss D, Wasil R, Metzger W. Capsular types and outcome of bacteremic pneumococcal disease in the antibiotic era. Arch Int Med 1974; 134:505-511  Back to cited text no. 1    
2.Nolan CM, Beaty HN, Bagdade JD. Further characterization of impaired bactericidal function of granulocytes in poorly controlled diabetes. Diabetes 1978; 27:889-893.  Back to cited text no. 2    
3.Rayfield EJ, Ault MJ, Keusch GT, Brothers MJ, Nechamias G, Smith H. Infection and diabetes: the case for glucose control. Am J Med 1982; 72:439-450.  Back to cited text no. 3    
4.Mowat AG, Baum J. Chemotaxis of polymorphonuclear leucocytes from patients with diabetes mellitus. N Engl J Med 1972; 284:621-627.  Back to cited text no. 4    
5.Bybee JD, Rogers DE. The phagocytic activity of polymorphonuclear leucocytes obtained from patients with diabetes mellitus. J Lab Clin Med 1964; 64:1-13.  Back to cited text no. 5    
6.Bagdade JD, Nielson KL, Bulger RJ. Reversible abnormalities in phagocytic function in poorly controlled diabetic patients. Am J Med Sci 1972; 263:451-456.  Back to cited text no. 6    
7.Bagdade JD, Root RK, Bulger RJ. Imparied leucocyte function in patients with poorly controlled diabetes. Diabetes 1974; 23:9-15.  Back to cited text no. 7    
8.Dubos RJ. Effect of ketone bodies and other metabolites on the survival and multiplication of staphylococci and tubercle bacilli. J Exp Med 1953; 98:145-155.  Back to cited text no. 8    
9.Aguado MT, Pujoi N, Rubiol E, Tura M, Celada A. Separation of granulocytes from peripheral blood in a single step using discontinuous density gradients of ficoll - urografin. A comparative study with separation by Dextran. J Immunol Meth 1980; 32:41-50.  Back to cited text no. 9    
10.Marcus PJ, Cieciura SJ, Puck JT. Clonal growth in vitro of epithelial cells from normal human tissue. J Exp Med 1956; 104:615-627.  Back to cited text no. 10    
11.Sawant JM, Taskar SP. Lysosomal enzyme release from normal human polymorphonuclear leucocytes in response to particulate stimuli. Ind J Clin Biochem 1991; 6:77-78.  Back to cited text no. 11    
12.Brittinger G, Hirschhorn R, Douglas Sd, Weissman G. Studies on lysosomes. X). Characterization of a hydrolase - rich fraction from human lymphocytes. J Cell Biol 1968; 37:394-411.  Back to cited text no. 12    
13.In: Freehold NJ, editor. Worthington Enzyme Manual. Worthington Biochemical Corporation, 1972:100-101.  Back to cited text no. 13    
14.Kind PRN, King EJ. Estimation of plasma phosphate by determination of hydrolyzed phenol with amino-antipyrine. J Clin Pathol 1954; 7:322-326.  Back to cited text no. 14    
15.King EJ, Abul-Fadl MAM, Walker PG. King Armstrong Phosphatase estimation by the determination of liberated phosphate. J Clin Pathol 1951; 4:85-91.  Back to cited text no. 15    
16.Wacker WEC, Ulmer DDT, Valler BL. Metalloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. N Engl J Med 1956; 255:449-456.  Back to cited text no. 16    
17.Weissmann G, Zurier RB, Spieler PJ, Goldstein IM. Mechanism of lysosomal enzyme release from leucocytes exposed to immune complexes and other particles. J Exp Med 1971; 134:149s-165s.  Back to cited text no. 17    
18.Davis AT, Estensen R, Quie PG. Cytochalasin B. III. Inhibition of human polymorphonuclear leucocytes phagocytosis. Proc Soc Exp Biol Med 1971; 137:161-164.  Back to cited text no. 18    
19.Zurier RB, Hoffstein S, Weissmann G, Cytochalasin B. Effect on lysosomal release from human leucocytes. Proc Natl Acad Sci 1973; 70:844-848.  Back to cited text no. 19    
20.Holmes, B, Page AR, Good RA. Studies of the metabolic activity of leucocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest 1967; 46:1422-1430.   Back to cited text no. 20    


[Table - 1], [Table - 2]

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Online since 12th February '04
2004 - Journal of Postgraduate Medicine
Official Publication of the Staff Society of the Seth GS Medical College and KEM Hospital, Mumbai, India
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