- Email: [email protected]
Hypoglycaemia and diabetes control
853
EDITORIALS
such as ascorbic acid and polyunsaturated fatty acids also undergo metal-catalysed oxidation and cause browning and fragmentation of proteins.’The exact role of glucose in some other plausible models of diabetic complications--eg, the widespread diabetesinduced reduction of glycosaminoglycans such as heparan sulphate8-is unknown. To reassess the hazards of modem optimised insulin treatment we
by asking whether intensified insulin regimens are invariably associated with an increased risk of hypoglycaemia compared with conventional may start
Hypoglycaemia and diabetes control Do the benefits of tight blood glucose control in diabetes warrant the risks? This issue was much discussed about fifteen years age1 when the development of methods for delivering insulin in a
physiological fashion was being strongly advocated,2 but has never been resolved satisfactorily. Many at that time were committed to the view that, if good control can be achieved, it would be beneficial and would lessen microvascular complications. Others pointed out that there were very real risks of increased hypoglycaemia with the "optimised" regimens then advocated, in addition to the psychosocial upsets and cost of intensive insulin therapy. The advent of home blood glucose monitoring, multiple insulin injection regimens, continuous subcutaneous insulin infusion, insulin pens, and easier more
of the average concentration of blood sugar via glycosylated haemoglobin and fructosamine have made long-term nearmeasurements normoglycaemia possible in many diabetic patients. However, this new experience engendered fresh concerns about the risks of improving diabetic control. Perhaps the most important worries are the discovery that improved control can itself impair release of catecholamines in response to low blood sugar3 and of thereby diminish warning symptoms more A far contentious hypoglycaemia. possibility is that a change from animal to human insulin may be associated in some patients with diminished autonomic warning symptoms and signs of low blood glucose levels and/or an increased frequency of assessment
hypoglycaemia.44 W olff5 points out that the increasing tendency to move hyperglycaemia from the status of risk factor to the main causal agent for diabetic tissue complications may be to focus too closely on glucose as the index of control. If metabolites other than or as well as glucose react with tissues in the pathogenesis of diabetic complications, should we strive (perhaps unsafely) for normoglycaemia with so little understanding of what we are trying ultimately to control (ie, complications)? Glucose is involved in the cross-linking and browning of proteins with a long half-life, either through reactions and rearrangements involving glycosylated adducts,6 or by transition metal-catalysed oxidation of glucose to protein-reactive ketoaldehydes, hydrogen peroxide, and reactive oxidants.Yet other molecules
insulin treatment that offers "standard" control. The on-going Diabetes Control and Complications Trial (DCCT) in North America is often taken as an example of how intensified control is linked with frequent hypoglycaemia.9 Patients with insulindependent diabetes (IDDM) are allocated to standard treatment with one or two injections of insulin a day or intensified treatment via an insulin pump or multiple daily injections. In the first 817 patients randomised in the DCCT, episodes of severe hypoglycaemia were about three-fold higher in the well-controlled group than in the standard group.9 A high rate of hypoglycaemia during strict control has been noted in other studies but most comparisons of conventional and optimised control show no significant differencell-14 or a lower rate of hypoglycaemia 15,16 with intensive treatment. Next, we must ask whether good control always leads to lowering of the glycaemic threshold for the release of catecholamines and impaired awareness of hypoglycaemia. Although it clearly did in some studies,3>1’ in others there was no change in the counterregulatory hormone response to insulininduced hypoglycaemia when strict control was compared with conventional contro1.18-20 Moreover, in one study good control achieved by continuous subcutaneous insulin infusion led to blunted adrenaline and cortisol secretion after hypoglycaemia, but the same average level of control achieved by intravenous insulin infusion in the same patients did not alter hormonal responses.21 So, intensive insulin therapy need not produce unacceptable hypoglycaemia and need not alter awareness of hypoglycaemia. What we do not know is whether such good results are achieved by patient selection (eg, exclusion of those predisposed to hypoglycaemia) or choice of a particular regimen (insulin delivery rate, education), or some other factor. Turning to human insulin, several reports have lately heightened continued controversy about a possible risk of hypoglycaemia in patients transferred from animal to human sequence insulin. In a casecontrol study, 23 diabetic patients admitted to hospital with severe hypoglycaemia were compared with those admitted for reasons other than hypoglycaemia. Treatment with human insulin was more common in the hypoglycaemic cases than in the nonhypoglycaemic controls (46% vs 34%) and the overall number of admissions for hypoglycaemia rose from
854
70/100000
160/100000
from 1984 to 1987, in parallel with the introduction of human insulin. In a randomised double-blind crossover trial from the same group,23 44 patients with IDDM were treated with either human or pork insulin for six weeks each. Blood glucose control was similar on the two treatments but a questionnaire of symptoms occurring during hypoglycaemia revealed that human insulin was more likely than porcine insulin to be associated with the neuroglycopenic features of lack of concentration, restlessness, and confusion and less likely to be linked with hunger (taken to be an autonomic symptom). However, the more definite autonomic symptoms of sweating and tremor did not differ between treatments. By contrast, three comparisons of intravenous pork and human insulin in IDDM24-26 have shown no important differences in hormonal responses to hypoglycaemia, even in patients who had previously reported loss of awareness on transfer to human insulin and later return of symptoms with animal insulin.26 In a study from Gennany, 27 247 patients on animal insulin were matched for duration of diabetes with 276 patients on human insulin. A questionnaire showed no differences in symptoms or frequency of severe hypoglycaemia in the two groups. Another questionnaire study,2g of 628 IDDM patients in Pittsburgh, provided no evidence of a substantially increased frequency of hypoglycaemia or reduced awareness thereof during human insulin treatment. In the DCCT, although the use of human insulin increased from 0 to 50% of subjects from 1983 to 1989, the overall rate of hypoglycaemia decreased over this period. In the Netherlands, hospital admission rates for severe hypoglycaemia decreased from 1983 to 1988 when the use of human insulin increased about 20-fold ;29 and in a prospective double blind trial in 1514 diabetic patients in the USA there were no differences in hypoglycaemia or symptoms.3o Thus no systematic difficulty has been shown with human insulin. Nevertheless, the reports of altered awareness from Switzerland,22,23 and also one from Australia,31 beg an explanation, especially when a large proportion of the patients were said to be affected.31 Again, variations in insulin delivery and/or control in different diabetic populations may be important-in some patients the otherwise trivial differences between human and pork insulin pharmacology may have been uncovered. The prevailing uncertainty about a link between hypoglycaemia unawareness and human insulin is reflected in this week’s announcement from the British Diabetic Association about a loss of warnings task force. The task force will establish a hypoglycaemia help line, prepare information for patients and doctors, and consider the need for further studies. Although strict metabolic control can probably be achieved in most IDDM patients without an unacceptable increase in hypoglycaemia or loss of warning, until the results of these latest deliberations to
available it seems wise in an individual patient to improve control gradually so the effects of a particular regimen can be defined. are
1.
2.
Siperstein MD, Foster DW, Knowles HC, Levine R, Madison LL, Roth J. Control of blood glucose and diabetic vascular disease. N Engl J Med 1977; 296: 1060-63. Cahill GF, Etzwiler DD, Freinkel N. "Control" and diabetes. N Engl J
Med 1976; 294: 1004-05. SA, Tamborlane WV, Simonson DC, Sherwin RS. Defective glucose counter-regulation after strict glycemic control of insulin dependent diabetes mellitus. N Engl J Med 1987; 316: 1376-83. 4. Teuscher A, Berger WB. Hypoglycaemia unawareness in diabetics transferred from beef/porcine insulin to human insulin. Lancet 1987 ii: 3. Amiel
382-85. 5. Wolff SP. Is hyperglycaemia risky enough to justify the increased risk of hypoglycaemia linked with tight diabetes control? Biochem Med Metab Biol 1991; 46: 129-38. 6. Dominiczak MH. The significance of the products of the Maillard (Browning) reaction in diabetes. Diabetic Med 1991; 8: 505-16. 7. Hunt JV, Wolff SP. Is glucose the sole source of tissue browning in diabetes mellitus? FEBS Lett 1990; 269: 258-60. 8. Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, Jensen T, KofoedEnevoldsen A. Albuminuria reflects widespread vascular damage: the Steno hypothesis. Diabetologia 1989; 32: 219-26. 9. DCCT Research Group. Epidemiology of severe hypoglycemia in the Diabetes Control and Complications Trial. Am J Med 1991; 90: 450-59. 10. Arias P, Kerner W, Zier H, Navascues I, Pfeiffer EF. Incidence of hypoglycemic episodes in diabetic patients under continuous subcutaneous insulin infusion and intensified conventional insulin treatment: assessment by means of semiambulatory 24-hour continuous blood glucose monitoring. Diabetes Care 1985; 8: 134-40. 11. Lauritzen T, Frost-Larsen K, Larsen H-W, et al. Effect of 1 year of near-normal blood glucose levels on retinopathy in insulin-dependent diabetes. Lancet 1983; i: 200-03. 12. Feldt-Rasmussen B, Mathiesen ER, Deckert T. Effect of two years of strict metabolic control on progression of incipient nephropathy in insulin-dependent diabetes. Lancet 1986; ii: 1300-04. 13. Helve E, Koivisto VA, Lehtonen A, Pelkonen R, Huttunen JK, Nikkila EA. A crossover comparison of continuous insulin infusion and conventional injection treatment of type 1 diabetes. Acta Med Scand 1987; 221: 385-93. 14. Mecklenberg RS, Benson EA, Benson JW, et al. Acute complications associated with insulin pump therapy. JAMA 1984; 252: 3265-69. 15. Bending JJ, Pickup JC, Keen H. Frequency of diabetic ketoacidosis and hypoglycemic coma during treatment with continuous subcutaneous insulin infusion. Am J Med 1985; 79: 685-91. 16. Eichner HL, Selam J-L, Holleman CB, Worcester BR, Turner DS, Charles MA. Reduction of severe hypoglycaemic events in type 1 (insulin dependent) diabetic patients using continuous subcutaneous insulin infusion. Diabetes Res 1988; 8: 189-93. 17. Lager I, Attvall S, Blohme G, Smith U. Altered recognition of hypoglycaemic symptoms in type 1 diabetes during intensified control with continuous subcutaneous insulin infusion. Diabetic Med 1986; 3: 322-25. 18. Bolli G, De Feo P, De Cosmo S, et al. Effects of long-term optimization and short-term deterioration of glycemic control on glucose counterregulation in type 1 diabetes mellitus. Diabetes 1984; 33: 394-400. 19. Lins PE, Adamson U, Kollind M, Hamberger B, Efendic S. Hormonal responses to insulin-induced hypoglycaemia after optimized glycaemic control in type 1 (insulin-dependent) diabetes. Diabetologia 1983; 25: 176A. 20. Ng Tang Fui S, Pickup JC, Bending JJ, et al. Hypoglycemia and couterregulation in insulin-dependent diabetic patients: a comparison of continuous subcutaneous insulin infusion and conventional insulin injection therapy. Diabetes Care 1986; 9: 221-27. 21. Gulan M, Perlman K, Sole M, Albisser AM, Zinman B. Counterregulatory hormone responses preserved after long-term intravenous insulin infusion compared to continuous subcutaneous insulin infusion. Diabetes 1988; 37: 526-31. 22. Egger M, Davey Smith G, Imhoof H, Teuscher A. Risk of severe hypoglycaemia in insulin treated diabetic patients transferred to human insulin: a case control study. Br Med J 1991; 303: 617-21. 23. Egger M, Davey Smith G, Teuscher AU, Teuscher A. Influence of human insulin on symptoms and awareness of hypoglycaemia: a randomised double blind trial. Br Med J 1991; 303: 622-26. 24. Bendtson I, Binder C. Counterregulatory hormonal response to insulininduced hypoglycaemia in insulin-dependent diabetic patients: a
855
comparison of equimolar amounts of porcine and semisynthetic human insulin. J Intern Med 1991; 229: 293-96. 25. Clausen Sjobom N, Lins P-E, Adamson U, Theodorsson E. A comparative study on the hormonal responses to insulin-induced hypoglycaemia using semisynthetic human insulin and pork insulin in patients with type 1 diabetes mellitus. Diabetic Med 1990; 7: 775-79. 26. Patrick AW, Bodmer CW, Tieszen KL, White MC, Williams G. Human insulin and awareness of acute hypoglycaemia in insulin-dependent diabetes. Lancet 1991; 338: 528-32. 27. Mühlhauser I, Heinemann L, Fritsche E, von Lenhep K, Berger M. Hypoglycemic symptoms and frequency of severe hypoglycemia in patients treated with human and animal insulin preparations. Diabetes Care 1991; 14: 745-49. 28. Orchard TJ, Maser RE, Becker DJ, Dorman JS, Drash AL. Human insulin use and hypoglycaemia: insights from the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetic Med 1991; 8: 469-74. 29. Heine RJ, van der Veen EA. Human insulin and hypoglycaemia. Lancet 1990; 335: 62. 30. Anderson JH, Holcomber JH, Grimes JA, Galloway JA. Hypoglycemia unawareness and human insulin. Diabetologia 1990; 33 (suppl): 122A. 31. Stocks AE. Human insulin. Med J Aust 1991. 154: 295-96.
Calprotectin, zinc, and abscesses Bacteria can survive, without proliferation, in an established abscess for many weeks unless the abscess is drained.1 Although neutrophils are plentiful, most are dead or disintegrating and abscess fluid can itself prevent neutrophil killing.2Other factors must inhibit but not kill bacteria and possibly render them less susceptible to bactericidal antibiotics. One candidate is the calcium-binding protein known as calprotectin or Ll protein that is found mainly in neutrophil cytoplasm and is stable in neutrophil lysates.3 This protein seems to act by binding zinc, an element that affects microbial growth and the host immune
response.4 Calprotectin
has a molecular weight of 36-5 kDa and is present in large amounts in human granulocytes and in normal mucosal squamous epithelium.5°6 There are three polypeptide chains in the protein-two heavy chains and a light chain--each of which can bind two calcium ions.’ Plasma concentrations are high in patients with bacterial infection, rising to 40-130 times normal in those with meningitis, septicaemia, and pneumonia.8 Calprotectin is also present in the epidermis of patients with psoriasis and allergic contact dermatitis.6The light chain seems to be identical to cystic fibrosis antigen (calgranulin), which is found in the serum of patients with the disease and in carriers of the cystic fibrosis gene.9 The light and heavy chains are the same as the MRP-8 and MRP-14 calcium-binding proteins derived from macrophages in chronic polyarthritis. 10 Portions of the aminoacid sequence of calprotectin are those of neutrophil-immobilising proteins (NIF-1 and NIF-2), so calprotectin might be important in the adherence of myeloid cells to the endothelium during the inflammatory response Release of an inhibitory factor from disrupted neutrophils but not from intact ones was shown by McNamara et al12, who studied growth of Candida albicans pseudohyphae. The same group 13 later found that the factor had a similar aminoacid sequence to calprotectin and incubation with a specific
antibody to calprotectin eliminated its inhibitory activity. A concentration of 21 mg/1 prevented the growth of 90% of strains of Candida albicans; at higher concentrations (288 and 77 mg/1), Staphylococcus aureus and Escherichia coli were inhibited. Steinbakk et al’ found minimum inhibitory concentrations of calprotectin of 4-32 mg/1 for C albicans, 256 mg/l for E coli and Klebsiella spp, 64 mg/1 for S aureus, and 64-256 mg/l for S epidermidis; killing was observed at concentrations 2-4 times the minimum inhibitory concentration. monoclonal
Sohnle et aP have now shown that the inhibition of C albicans growth by human neutrophil lysates or abscess fluid supernatant is reversed by a low concentration of zinc (1 -4-14 pmol/1); other trace elements, including iron, were ineffective. Addition of neutrophil lysate protein prevented zinc passing through a dialysis membrane. The presence of calprotectin was confirmed by application of monoclonal antibody to western blots of fluid. human abscess electrophoresis gels containing However, human abscess fluid protein only inhibited and did not kill C albicans. Calprotectin released from dead neutrophils seemed to exert its antifungal effect by depriving the yeast of zinc, but some supporting data were not presented and effects on bacteria were not examined. Most zinc ingested in the diet is lost in the faeces but a serum concentration of 1 mg/1 is maintained, largely in a bound form; concentrations fall by 10-60% in infections and other acute illnesses.4 The metal probably stabilises cellular membranes; neutrophil phagocytosis is inhibited in the physiological range. In bacteria, zinc is needed for functioning of certain enzymes (eg, nucleic acid polymerases), and for production of virulence factors.4Apart from a few yeasts, microorganisms have no mechanism for sequestering zinc and might be susceptible to zinc deprivation. However, bacteria require only very low concentrations (10-4 to 10-2 mmol/1) for growth, so in-vitro study of zinc deficiency is difficult. Bacteria are inhibited by zinc concentrations of 0.5-32 mmol/1 and zinc-impregnated dressings are widely used to reduce bacterial counts in wounds and promote healing.14 Thus host defences might inhibit bacterial growth in pus by reducing available zinc whereas medicine achieves the same end by raising it. We do not know whether calprotectin is a sufficiently powerful chelator of zinc to inhibit bacteria in this way, but it is remarkable that only now is the function of one of the most abundant proteins in pus becoming understood. Joiner KA, Onderdonk AB, Gelfand JA, Bartlett JG, Gorbach SL. A quantitative model for subcutaneous abscess formation in mice. Br J Exp Pathol 1980; 61: 97-107. 2. Bamberger DM, Herndon BL. Bactericidal capacity of neutrophils in rabbits with experimental acute and chronic abscesses. J Infect Dis 1.
1990; 162: 186-92. 3. Sohnle PG, Collins-Lech C, Wiessner JH. The zinc-reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J Infect Dis 1991; 164: 137-42. 4. Sugarman B. Zinc and infection. Rev Infect Dis 1983; 5: 137-47.
EDITORIALS
such as ascorbic acid and polyunsaturated fatty acids also undergo metal-catalysed oxidation and cause browning and fragmentation of proteins.’The exact role of glucose in some other plausible models of diabetic complications--eg, the widespread diabetesinduced reduction of glycosaminoglycans such as heparan sulphate8-is unknown. To reassess the hazards of modem optimised insulin treatment we
by asking whether intensified insulin regimens are invariably associated with an increased risk of hypoglycaemia compared with conventional may start
Hypoglycaemia and diabetes control Do the benefits of tight blood glucose control in diabetes warrant the risks? This issue was much discussed about fifteen years age1 when the development of methods for delivering insulin in a
physiological fashion was being strongly advocated,2 but has never been resolved satisfactorily. Many at that time were committed to the view that, if good control can be achieved, it would be beneficial and would lessen microvascular complications. Others pointed out that there were very real risks of increased hypoglycaemia with the "optimised" regimens then advocated, in addition to the psychosocial upsets and cost of intensive insulin therapy. The advent of home blood glucose monitoring, multiple insulin injection regimens, continuous subcutaneous insulin infusion, insulin pens, and easier more
of the average concentration of blood sugar via glycosylated haemoglobin and fructosamine have made long-term nearmeasurements normoglycaemia possible in many diabetic patients. However, this new experience engendered fresh concerns about the risks of improving diabetic control. Perhaps the most important worries are the discovery that improved control can itself impair release of catecholamines in response to low blood sugar3 and of thereby diminish warning symptoms more A far contentious hypoglycaemia. possibility is that a change from animal to human insulin may be associated in some patients with diminished autonomic warning symptoms and signs of low blood glucose levels and/or an increased frequency of assessment
hypoglycaemia.44 W olff5 points out that the increasing tendency to move hyperglycaemia from the status of risk factor to the main causal agent for diabetic tissue complications may be to focus too closely on glucose as the index of control. If metabolites other than or as well as glucose react with tissues in the pathogenesis of diabetic complications, should we strive (perhaps unsafely) for normoglycaemia with so little understanding of what we are trying ultimately to control (ie, complications)? Glucose is involved in the cross-linking and browning of proteins with a long half-life, either through reactions and rearrangements involving glycosylated adducts,6 or by transition metal-catalysed oxidation of glucose to protein-reactive ketoaldehydes, hydrogen peroxide, and reactive oxidants.Yet other molecules
insulin treatment that offers "standard" control. The on-going Diabetes Control and Complications Trial (DCCT) in North America is often taken as an example of how intensified control is linked with frequent hypoglycaemia.9 Patients with insulindependent diabetes (IDDM) are allocated to standard treatment with one or two injections of insulin a day or intensified treatment via an insulin pump or multiple daily injections. In the first 817 patients randomised in the DCCT, episodes of severe hypoglycaemia were about three-fold higher in the well-controlled group than in the standard group.9 A high rate of hypoglycaemia during strict control has been noted in other studies but most comparisons of conventional and optimised control show no significant differencell-14 or a lower rate of hypoglycaemia 15,16 with intensive treatment. Next, we must ask whether good control always leads to lowering of the glycaemic threshold for the release of catecholamines and impaired awareness of hypoglycaemia. Although it clearly did in some studies,3>1’ in others there was no change in the counterregulatory hormone response to insulininduced hypoglycaemia when strict control was compared with conventional contro1.18-20 Moreover, in one study good control achieved by continuous subcutaneous insulin infusion led to blunted adrenaline and cortisol secretion after hypoglycaemia, but the same average level of control achieved by intravenous insulin infusion in the same patients did not alter hormonal responses.21 So, intensive insulin therapy need not produce unacceptable hypoglycaemia and need not alter awareness of hypoglycaemia. What we do not know is whether such good results are achieved by patient selection (eg, exclusion of those predisposed to hypoglycaemia) or choice of a particular regimen (insulin delivery rate, education), or some other factor. Turning to human insulin, several reports have lately heightened continued controversy about a possible risk of hypoglycaemia in patients transferred from animal to human sequence insulin. In a casecontrol study, 23 diabetic patients admitted to hospital with severe hypoglycaemia were compared with those admitted for reasons other than hypoglycaemia. Treatment with human insulin was more common in the hypoglycaemic cases than in the nonhypoglycaemic controls (46% vs 34%) and the overall number of admissions for hypoglycaemia rose from
854
70/100000
160/100000
from 1984 to 1987, in parallel with the introduction of human insulin. In a randomised double-blind crossover trial from the same group,23 44 patients with IDDM were treated with either human or pork insulin for six weeks each. Blood glucose control was similar on the two treatments but a questionnaire of symptoms occurring during hypoglycaemia revealed that human insulin was more likely than porcine insulin to be associated with the neuroglycopenic features of lack of concentration, restlessness, and confusion and less likely to be linked with hunger (taken to be an autonomic symptom). However, the more definite autonomic symptoms of sweating and tremor did not differ between treatments. By contrast, three comparisons of intravenous pork and human insulin in IDDM24-26 have shown no important differences in hormonal responses to hypoglycaemia, even in patients who had previously reported loss of awareness on transfer to human insulin and later return of symptoms with animal insulin.26 In a study from Gennany, 27 247 patients on animal insulin were matched for duration of diabetes with 276 patients on human insulin. A questionnaire showed no differences in symptoms or frequency of severe hypoglycaemia in the two groups. Another questionnaire study,2g of 628 IDDM patients in Pittsburgh, provided no evidence of a substantially increased frequency of hypoglycaemia or reduced awareness thereof during human insulin treatment. In the DCCT, although the use of human insulin increased from 0 to 50% of subjects from 1983 to 1989, the overall rate of hypoglycaemia decreased over this period. In the Netherlands, hospital admission rates for severe hypoglycaemia decreased from 1983 to 1988 when the use of human insulin increased about 20-fold ;29 and in a prospective double blind trial in 1514 diabetic patients in the USA there were no differences in hypoglycaemia or symptoms.3o Thus no systematic difficulty has been shown with human insulin. Nevertheless, the reports of altered awareness from Switzerland,22,23 and also one from Australia,31 beg an explanation, especially when a large proportion of the patients were said to be affected.31 Again, variations in insulin delivery and/or control in different diabetic populations may be important-in some patients the otherwise trivial differences between human and pork insulin pharmacology may have been uncovered. The prevailing uncertainty about a link between hypoglycaemia unawareness and human insulin is reflected in this week’s announcement from the British Diabetic Association about a loss of warnings task force. The task force will establish a hypoglycaemia help line, prepare information for patients and doctors, and consider the need for further studies. Although strict metabolic control can probably be achieved in most IDDM patients without an unacceptable increase in hypoglycaemia or loss of warning, until the results of these latest deliberations to
available it seems wise in an individual patient to improve control gradually so the effects of a particular regimen can be defined. are
1.
2.
Siperstein MD, Foster DW, Knowles HC, Levine R, Madison LL, Roth J. Control of blood glucose and diabetic vascular disease. N Engl J Med 1977; 296: 1060-63. Cahill GF, Etzwiler DD, Freinkel N. "Control" and diabetes. N Engl J
Med 1976; 294: 1004-05. SA, Tamborlane WV, Simonson DC, Sherwin RS. Defective glucose counter-regulation after strict glycemic control of insulin dependent diabetes mellitus. N Engl J Med 1987; 316: 1376-83. 4. Teuscher A, Berger WB. Hypoglycaemia unawareness in diabetics transferred from beef/porcine insulin to human insulin. Lancet 1987 ii: 3. Amiel
382-85. 5. Wolff SP. Is hyperglycaemia risky enough to justify the increased risk of hypoglycaemia linked with tight diabetes control? Biochem Med Metab Biol 1991; 46: 129-38. 6. Dominiczak MH. The significance of the products of the Maillard (Browning) reaction in diabetes. Diabetic Med 1991; 8: 505-16. 7. Hunt JV, Wolff SP. Is glucose the sole source of tissue browning in diabetes mellitus? FEBS Lett 1990; 269: 258-60. 8. Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, Jensen T, KofoedEnevoldsen A. Albuminuria reflects widespread vascular damage: the Steno hypothesis. Diabetologia 1989; 32: 219-26. 9. DCCT Research Group. Epidemiology of severe hypoglycemia in the Diabetes Control and Complications Trial. Am J Med 1991; 90: 450-59. 10. Arias P, Kerner W, Zier H, Navascues I, Pfeiffer EF. Incidence of hypoglycemic episodes in diabetic patients under continuous subcutaneous insulin infusion and intensified conventional insulin treatment: assessment by means of semiambulatory 24-hour continuous blood glucose monitoring. Diabetes Care 1985; 8: 134-40. 11. Lauritzen T, Frost-Larsen K, Larsen H-W, et al. Effect of 1 year of near-normal blood glucose levels on retinopathy in insulin-dependent diabetes. Lancet 1983; i: 200-03. 12. Feldt-Rasmussen B, Mathiesen ER, Deckert T. Effect of two years of strict metabolic control on progression of incipient nephropathy in insulin-dependent diabetes. Lancet 1986; ii: 1300-04. 13. Helve E, Koivisto VA, Lehtonen A, Pelkonen R, Huttunen JK, Nikkila EA. A crossover comparison of continuous insulin infusion and conventional injection treatment of type 1 diabetes. Acta Med Scand 1987; 221: 385-93. 14. Mecklenberg RS, Benson EA, Benson JW, et al. Acute complications associated with insulin pump therapy. JAMA 1984; 252: 3265-69. 15. Bending JJ, Pickup JC, Keen H. Frequency of diabetic ketoacidosis and hypoglycemic coma during treatment with continuous subcutaneous insulin infusion. Am J Med 1985; 79: 685-91. 16. Eichner HL, Selam J-L, Holleman CB, Worcester BR, Turner DS, Charles MA. Reduction of severe hypoglycaemic events in type 1 (insulin dependent) diabetic patients using continuous subcutaneous insulin infusion. Diabetes Res 1988; 8: 189-93. 17. Lager I, Attvall S, Blohme G, Smith U. Altered recognition of hypoglycaemic symptoms in type 1 diabetes during intensified control with continuous subcutaneous insulin infusion. Diabetic Med 1986; 3: 322-25. 18. Bolli G, De Feo P, De Cosmo S, et al. Effects of long-term optimization and short-term deterioration of glycemic control on glucose counterregulation in type 1 diabetes mellitus. Diabetes 1984; 33: 394-400. 19. Lins PE, Adamson U, Kollind M, Hamberger B, Efendic S. Hormonal responses to insulin-induced hypoglycaemia after optimized glycaemic control in type 1 (insulin-dependent) diabetes. Diabetologia 1983; 25: 176A. 20. Ng Tang Fui S, Pickup JC, Bending JJ, et al. Hypoglycemia and couterregulation in insulin-dependent diabetic patients: a comparison of continuous subcutaneous insulin infusion and conventional insulin injection therapy. Diabetes Care 1986; 9: 221-27. 21. Gulan M, Perlman K, Sole M, Albisser AM, Zinman B. Counterregulatory hormone responses preserved after long-term intravenous insulin infusion compared to continuous subcutaneous insulin infusion. Diabetes 1988; 37: 526-31. 22. Egger M, Davey Smith G, Imhoof H, Teuscher A. Risk of severe hypoglycaemia in insulin treated diabetic patients transferred to human insulin: a case control study. Br Med J 1991; 303: 617-21. 23. Egger M, Davey Smith G, Teuscher AU, Teuscher A. Influence of human insulin on symptoms and awareness of hypoglycaemia: a randomised double blind trial. Br Med J 1991; 303: 622-26. 24. Bendtson I, Binder C. Counterregulatory hormonal response to insulininduced hypoglycaemia in insulin-dependent diabetic patients: a
855
comparison of equimolar amounts of porcine and semisynthetic human insulin. J Intern Med 1991; 229: 293-96. 25. Clausen Sjobom N, Lins P-E, Adamson U, Theodorsson E. A comparative study on the hormonal responses to insulin-induced hypoglycaemia using semisynthetic human insulin and pork insulin in patients with type 1 diabetes mellitus. Diabetic Med 1990; 7: 775-79. 26. Patrick AW, Bodmer CW, Tieszen KL, White MC, Williams G. Human insulin and awareness of acute hypoglycaemia in insulin-dependent diabetes. Lancet 1991; 338: 528-32. 27. Mühlhauser I, Heinemann L, Fritsche E, von Lenhep K, Berger M. Hypoglycemic symptoms and frequency of severe hypoglycemia in patients treated with human and animal insulin preparations. Diabetes Care 1991; 14: 745-49. 28. Orchard TJ, Maser RE, Becker DJ, Dorman JS, Drash AL. Human insulin use and hypoglycaemia: insights from the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetic Med 1991; 8: 469-74. 29. Heine RJ, van der Veen EA. Human insulin and hypoglycaemia. Lancet 1990; 335: 62. 30. Anderson JH, Holcomber JH, Grimes JA, Galloway JA. Hypoglycemia unawareness and human insulin. Diabetologia 1990; 33 (suppl): 122A. 31. Stocks AE. Human insulin. Med J Aust 1991. 154: 295-96.
Calprotectin, zinc, and abscesses Bacteria can survive, without proliferation, in an established abscess for many weeks unless the abscess is drained.1 Although neutrophils are plentiful, most are dead or disintegrating and abscess fluid can itself prevent neutrophil killing.2Other factors must inhibit but not kill bacteria and possibly render them less susceptible to bactericidal antibiotics. One candidate is the calcium-binding protein known as calprotectin or Ll protein that is found mainly in neutrophil cytoplasm and is stable in neutrophil lysates.3 This protein seems to act by binding zinc, an element that affects microbial growth and the host immune
response.4 Calprotectin
has a molecular weight of 36-5 kDa and is present in large amounts in human granulocytes and in normal mucosal squamous epithelium.5°6 There are three polypeptide chains in the protein-two heavy chains and a light chain--each of which can bind two calcium ions.’ Plasma concentrations are high in patients with bacterial infection, rising to 40-130 times normal in those with meningitis, septicaemia, and pneumonia.8 Calprotectin is also present in the epidermis of patients with psoriasis and allergic contact dermatitis.6The light chain seems to be identical to cystic fibrosis antigen (calgranulin), which is found in the serum of patients with the disease and in carriers of the cystic fibrosis gene.9 The light and heavy chains are the same as the MRP-8 and MRP-14 calcium-binding proteins derived from macrophages in chronic polyarthritis. 10 Portions of the aminoacid sequence of calprotectin are those of neutrophil-immobilising proteins (NIF-1 and NIF-2), so calprotectin might be important in the adherence of myeloid cells to the endothelium during the inflammatory response Release of an inhibitory factor from disrupted neutrophils but not from intact ones was shown by McNamara et al12, who studied growth of Candida albicans pseudohyphae. The same group 13 later found that the factor had a similar aminoacid sequence to calprotectin and incubation with a specific
antibody to calprotectin eliminated its inhibitory activity. A concentration of 21 mg/1 prevented the growth of 90% of strains of Candida albicans; at higher concentrations (288 and 77 mg/1), Staphylococcus aureus and Escherichia coli were inhibited. Steinbakk et al’ found minimum inhibitory concentrations of calprotectin of 4-32 mg/1 for C albicans, 256 mg/l for E coli and Klebsiella spp, 64 mg/1 for S aureus, and 64-256 mg/l for S epidermidis; killing was observed at concentrations 2-4 times the minimum inhibitory concentration. monoclonal
Sohnle et aP have now shown that the inhibition of C albicans growth by human neutrophil lysates or abscess fluid supernatant is reversed by a low concentration of zinc (1 -4-14 pmol/1); other trace elements, including iron, were ineffective. Addition of neutrophil lysate protein prevented zinc passing through a dialysis membrane. The presence of calprotectin was confirmed by application of monoclonal antibody to western blots of fluid. human abscess electrophoresis gels containing However, human abscess fluid protein only inhibited and did not kill C albicans. Calprotectin released from dead neutrophils seemed to exert its antifungal effect by depriving the yeast of zinc, but some supporting data were not presented and effects on bacteria were not examined. Most zinc ingested in the diet is lost in the faeces but a serum concentration of 1 mg/1 is maintained, largely in a bound form; concentrations fall by 10-60% in infections and other acute illnesses.4 The metal probably stabilises cellular membranes; neutrophil phagocytosis is inhibited in the physiological range. In bacteria, zinc is needed for functioning of certain enzymes (eg, nucleic acid polymerases), and for production of virulence factors.4Apart from a few yeasts, microorganisms have no mechanism for sequestering zinc and might be susceptible to zinc deprivation. However, bacteria require only very low concentrations (10-4 to 10-2 mmol/1) for growth, so in-vitro study of zinc deficiency is difficult. Bacteria are inhibited by zinc concentrations of 0.5-32 mmol/1 and zinc-impregnated dressings are widely used to reduce bacterial counts in wounds and promote healing.14 Thus host defences might inhibit bacterial growth in pus by reducing available zinc whereas medicine achieves the same end by raising it. We do not know whether calprotectin is a sufficiently powerful chelator of zinc to inhibit bacteria in this way, but it is remarkable that only now is the function of one of the most abundant proteins in pus becoming understood. Joiner KA, Onderdonk AB, Gelfand JA, Bartlett JG, Gorbach SL. A quantitative model for subcutaneous abscess formation in mice. Br J Exp Pathol 1980; 61: 97-107. 2. Bamberger DM, Herndon BL. Bactericidal capacity of neutrophils in rabbits with experimental acute and chronic abscesses. J Infect Dis 1.
1990; 162: 186-92. 3. Sohnle PG, Collins-Lech C, Wiessner JH. The zinc-reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J Infect Dis 1991; 164: 137-42. 4. Sugarman B. Zinc and infection. Rev Infect Dis 1983; 5: 137-47.