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Increased prostaglandin synthesis in childhood diabetes mellitus
February 1979 The Journal o f P E D I A T R I C S
185
Increased prostaglandin synthesis in childhood diabetes mellitus Prostaglandins are synthesized from the fatty acids, linoleic and arachidonic acids, and are associated with increased platelet aggregation as has been found in blood from patients with diabetes mellitus. In the present study blood was obtained from 40 children with diabetes and from 20 control children for measurements of fatty acid and PGE1, PGE.,_, and PGF.,a levels. The production of PGE., and PGF~a was significantly elevated in blood from the children with diabetes at all times measured. The mean quantitative plasma linoleic acid levels were also higher in the patients. The increased PG synthesis may be related to the vascular problems that occur in patients with diabetes.
H. Peter Chase, M.D,* Ricky L. Williams, M.D., Jaequeline Dupont, Ph.D.,
D e n v e r , Colo.,
and
F o r t Collins, Colo.
PROSTAGLANDINS are synthesized in almost all body tissues, including in platelets during blood clotting? Inflammation ~-and enhancement of platelet coagulation :~ are among the many physiologic effects of PGs, and both may be important in the vascular complications of diabetes. In vitro platelet aggregation time has been shown to be shorter in persons with diabetes than in normal persons? -1~ This effect may be secondary to increased PG production. In a recent report, increased production of a "PGE-like" material during platelet aggregation in adults with diabetes compared to controls was described? Platelet aggregation time in adults with diabetes is increased toward normal following the ingestion of aspirin, 7 a known inhibitor of PG synthesis. Fatty acids, including linoleic acid which is a substrate for PG synthesis (Figure), have been described to be altered in quantity in adults with diabetes. H. 12 In the present study, the net synthesis of PGE1, PGE2, and PGF~a was measured in whole blood from control children and From the University of Colorado Medical Center, Department of Pediatrics, and Colorado State University, Department of Food and Nutrition. Supported by Public Health Service Clinical Research Center Grant RR-69, by a gift from the American Diabetes Association, Fort Collins Unit, and by a USDA, C S R S Special Project Grant. *Reprint address: Universityof Colorado Medical Center, Department of Pediatrics, 4200 E. Ninth A re., Denver, CO 80262.
0022-3476/79/200185 +05500.50/0 9 1979 The C. V. Mosby Co.
children with diabetes. All Possible regulators of PG synthesis are present in whole blood, and the use of whole blood in production of PGs during clotting is considered to represent a physiologic biopsy technique. The levels of plasma and red blood cell FAs were also measured to determine their contribution or relation to PG production.
METHODS Forty children with insulin-dependent diabetes mellitus and 20 control children had blood drawn from the antecubital vein following an 1i- to 12-hour fast. All control and diabetic children were between the ages of 3 and 19 years, with both groups having similar ag e and sex distributions. Informed consent was obtained from the parents of all test and control children. All o f the test children were known to have had diabetes for at least one year and were otherwise well at the time of testing. Morning insulin injections were not given until after venipuncture. Prostaglandins. Net synthesis of PGE1, PGE,, and PGF2a by whole blood during clotting at 37~ was determined 10, 20, and 70 minutes following venipuncture. Synthesis was stopped by removal of blood cells by centrifugation, and the resulting serum was frozen for later radioimmunoassay?3 Sera from control and diabetic children were assayed simultaneously, with the assayer (J.D.) unaware of which group the samples were from. Fatty acids. Fatty acids were determined by a modifi-
Vol. 94, No. 2, pp. 185-189
1 86
Chase, Williams, and Dupont
The Journal of Pediatrics Febrtr 1979
"Desaturation Pathway" ~linolenic acid (18:3A6,9,12)
to other prostaglandins includlng PGa 2 and PGF2a
to: PGE 1 and PGFIa
6-de saturase /
LinoleLc Acid (18:2~9,12)
Elongation enzyme
T
.T/
Dihomogmmalinoleic
-.....
/
acid
~_ A r a c h i d o n i c Acid (20:4A5,8,11,14)
(20:3A8,11,14)
Eicosadlenoic Acid
(20:2611,14)
"~"'"~Eicosatrienoic Acid (dead end) (20:3A5,11,14)
"Elongation Pathway" (minor)
Figure. Metabolism of linoleic acid to arachidonic acid and prostaglandins. Other products of arachidonic acid, including endoperoxides and newly diagnosed PGs, have been reviewed recently.-'" cation of a previously described method." Plasma was separated from the RBCs, which were then washed three times with 0.9% saline. The FAs were extracted from the plasma and the RBCs with 2:1 chloroform:methanol containing 0.005% butylated hydroxytoluene as antioxidant. The RBC extracts were heated in a 60~ water bath for one hour and then both the plasma and RBC extracts were shaken mechanically for 30 minutes to obtain maximal extraction of FAs. Samples were methylated by heating at 60 ~ for 30 minutes with boron trifluoride in methanol. Fatty acid methyl esters were then extracted with petroleum ether, concentrated, and measured using a Hewlett-Packard gas chromatograph with automatic integrator computer. A coiled glass column, 180 cm x 2 mm, packed with I0% SP 2340 on 100/120 mesh Chromosorb W AW (Supelco) was used. Identification was made by comparison to pure standards and quantification was achieved by recovery of a C15 internal Standard~ Total FAs were calculated as the sum of individual values. All extractions were completed within 48 hours of venipuncture. Phospholipids were purified following the initial chloroform:methanol extraction (above) using Unisil column chromatography. '5 Preliminary experiments demonstrated that 99% of radioactivity was recovered following Unisil column chromatography. After column purification, the samples were methylated and analyzed on the gas chromatograph as above. Measures of diabetic control. All children with diabetes were asked to bring in a 24-hour urine specimen at the time of blood testing to measure glucose spillage as an index of glycemic control. Creatinine measurements to verify that the sample was a 24-hour collection were not made, but in most cases the children had parental supervision during the collection, and most were already
familiar with the technique. Urine and serum glucose levels were measured by the glucose oxidase method. ''~ Blood was obtained from the children with diabetes for fasting serum glucose levels and for cholesterol and triglyceride levels, as previously described, '7 when the samples were drawn for PG and FA analyses. Statistical analyses. Differences between the patient and control groups were tested using the Student t test, for which P values are given. Correlation coefficients for the values of children with diabetes were computed using the Pearson formula, and r values are included. RESULTS
Prostaglandin levels. Serum PGE1, PGE~, and PGF~a values for 20 control children and 40 children with diabetes are shown in the Table. Prostaglandin E., and PGF~a levels were significantly greater in diabetic children compared to controls at all three incubation times measured, i.e., 10, 20, and 70 minutes. Prostaglandin E1 values were significantly higher in the test children only at 20 minutes. The most frequently elevated PGs (above 2 SD of normal at any of the three times studied) in the children with diabetes were PGE~ (17 children) and PGFz a (19 children). Ten children had elevation of PGE, at any of the three times. Some children who had elevated PG levels at one incubation time did not have elevated levels at a different incubation time. The number of children with elevations at each incubation time is shown in parentheses in the Table. Three children had elevations ( > 2 SD of normal) of all 3 PGs (at one or more of the three time periods), whereas four children did not have elevations of any PG at any time measured. Including the three children who had elevations of all three PGs, five had elevations (at any of
Volume 94 Number 2
the times measured ) of both PGE, and PGE~, i l had elevations of PGE~ and PGF2a, and six had elevations of PGE1 and PGF2a. Prostaglandin levels did not significantly (P > 0.05) correlate with age, sex, or duration of diabetes in the diabetic children. Further analyses were done using the Student t test, in which the children with diabetes were divided into those with: (1) an elevated level of one of the three PGs (at any of the three time periods), or (2) a normal level (at all time periods) of the same PG. There were again no significant (P > 0.05) differences between the two groups for age, sex, or duration of diabetes for any of the three PGs. Linoleic acid levels were significantly higher (P < 0.05) in the group with elevated PGE~ values, and cholesterol levels were higher in those with elevated PGE1 (P < 0.01), compared to the values for diabetic children with normal levels of the corresponding PG. Other comparisons between the two groups were not significantly different (P > 0.05). Plasma fatty acids. The percentages of individual FAs in plasma from the diabetic and the control children were similar except for stearic acid (18:0), which was higher (P < 0.01) in the 20 control children (11.5 _ 2.3%; mean ___1 SD) than in the 40 diabetic children (9.9 _ 1.4%). When FAs were expressed quantitatively, in mg/dl, palmitic acid (16:0) was significantly higher (P < 0.05) in the children with diabetes ( 4 7 . 1 _ 10.4) than in the control children (42.6 _+ 6.6), as were oleic acid (18: 1A9) (diabetic children: 45.2 _ 15.5 vs controls: 36.5 -4- 7.3; P < 0.01) and linoleic acid (18:2A9,12) (diabetic children: 60.0 _+ 17.2 vs controls: 51.5 +__ 13.5; P < 0.05). Total FAs, expressed in mg/dl, were also higher (P < 0.01) in the 40 children with diabetes (200.2 ___ 45.2) as compared to the 20 control children (174.8 _ 27.0). Plasma phospholipid FAs were determined on the first 15 patients and the first 11 control children, and as there were no significant differences (P > 0.05) between the two groups of children, no further analyses were done. RBC fatty acids. The percentage and quantitative levels of RBC FAs were similar for the control children and the children with diabetes for all FAs except palmitoleic acid (16:1A7) (test children: 0.9 _+ 0.5% vs control children: 1.9 _+ 1.5%; P < 0.01). Since it was apparent after analysis of RBC FAs on the first 15 children with diabetes that values were very similar to those of control children, further samples for RBC FAs were not obtained. There were no significant differences (P > 0.05) in RBC phospholipids between the first 11 control and the first 15 test children.
Prostaglandin: fatty acid correlations for the children with diabetes. Prostaglandin E~ levels (at all three times measured) correlated positively with mg of plasma linole-
Prostaglandin synthesis in diabetes mellitus
18 7
Table. Prostaglandin production in the blood of control children and of children with diabetes*
Incubation time
PGE1 Control children Diabetic children
PGE., Control children Diabetic children
PGF~a Control children Diabetic children
1Omen
[ 20rain
] 70 min
1.22 -+ 1.36 2.82 +4.41 (5)
2.72 _+2.47 6.17 -+6.65t (9)
7.01 -+3.17 9.65 +8.22 (5)
0.33 --+0.74 1.56 _ 1.941" (6)
1.31 -4-1.60 4.22 -- 3.975 (9)
2.49 ' -+ 1.16 5.92 -+4.00w (15)
0.20 -- 0.77 1.08 _+1.70t (6)
0.81 -----1.50 3.76 --+3.90w (9)
1.85 - 1.21 6.96 _+4.73w (19)
*Results are expressed in ng/ml and represent the mean + 1 SD for 20 control children and 40 diabetic children. The number of children with elevations (>2 SD of normal) at each of the time periods is shown in parentheses. tP < 0.05. ~P < 0.01. w < 0.001. ic acid (P < 0.05, r = 0.36), and correlated with mg of oleic acid (P < 0.05, r = 0.35) and total fatty acids (P < 0.05, r = 0.35) at 70 minutes. Prostaglandin E~ levels correlated with mg of plasma linoleic acid (P < 0.05, r = 0.37), oleic acid (P < 0.05, r = 0.36) and total FAs (P < 0.05, r = 0.34) at 70 minutes. No other FAs correlated significantly with PGE, or PGE~ levels, and PGF~ a levels did not correlate with levels of any FA.
Measures of diabetic control (glucose, triglyceride, cholesterol). Serum glucose levels were < 150 m g / d l in 11 of the patients, from 151 to 299 m g / d l in 23 children, and > 3 0 0 mg/dl in six children. Serum glucose levels did not correlate significantly with specific FA or P G values, or with cholesterol and triglyceride levels. Urine glucose levels were < 5 0 gm/24 hours in 24 patients, between 50 and 100 gm/24 hours in eight patients, and > 100 gm/24 hours in eight children. Urine glucose spillage correlated with serum glucose levels at P < 0.001 (r = 0.49). Total FA levels in patients who spilled > 5 0 gm glucose were 215.4 mg/dl, compared to 190.1 mg/dl (P = 0.08) in those spilling < 5 0 gm. Urine glucose spillage did not correlate significantly with levels of specific FAs or PGs.
18 8
Chase, Williams, and Dupont
Serum triglyceride values were normal ( < 130 mg/dl) 17 in 22 children, between 130 and 200 mg/dl in 10, and >200 mg/dl in eight children. Total FA values were 213.3 mg/dl in children with triglyceride levels > 130 mg/dl, Compared to 190.6 mg/dl in those with levels < 130 mg/dl (P = 0.11). The percentages of palmitic and stearic acids, but not of other FAs, were significantly higher (P < 0.05) for children with triglyceride levels > 130 mg/dl than in children with values < 130 mg/dl. Triglyceride values did not correlate significantly with specific PG levels. Serum cholesterol levels were in the normal range (<210 mg/dl)" in 30 children; 10 children had values that were >210 mg/dl. Six of the 10 children had both elevated serum cholesterol and triglyceride values. Cholesterol values correlated with levels of palmitic (P < 0.05, r = 0.29), palmitoleic (P < 0.02, r = 0.35), stearic (P < 0.002, r = 0.44), linoleic (P < 0.02, r = 0.36) and dihomogammalinolenic (20:32x5,8,11) ( P < 0 . 0 5 , r = 0.27) acids. Cholesterol levels also correlated with PGE, levels at 20 minutes (P < 0.005, r = 0.52) and at 70 minutes (P < 0.05, r = 0.37) incubation. DISCUSSION Mean PGE2 and PGF2a production was significantly elevated in blood from children with diabetes at all times studied (Table). Linoieic acid is currently believed to be important in regulating PG synthesis and was significantly higher in the Plasma of Children with diabetes compared to control children. Linoleic acid is the dietary source of dihomogammalinolenic acid and arachidonic acid, which are the substrates for PG synthesis (Figure). A deficiency of linoleic acid in the diet has been shown to result in decreased PG synthesis, whereas an excess of dietary linoleic acid caused increased PG synthesis during blood dotting.~8, a9 In the present study, linoleic acid levels correlated significantly with PGE~ values at all three times measured, and with PGE2 values at 70 minutes in the children with diabetes. However, it is likely that other factors were also involved in the elevated PG production. All three PGs should have been similarly elevated if Substrate alone was the cause of increased PG synthesis, but elevations of the two-series of PGs were found more frequently than of the one-series. Current knowledge of PG metabolism indicates that ratios of various PG and thromboxane compounds are important. ~~ Regulation of the synthesis of PGs involves oxygen, glutathione, peroxidases, vitamin E, selenium, and levels of fatty acids. '~1 Future studies should be aimed at determining whether manipulation of the ratios, inhibition of synthesis, or altering other factors regulating PG synthesis might have an effect on decreasing the vascular complications of diabetes.
The Journal of Pediatrics February 1979
There were no statistically significant correlations between PG levels and urine or serum glucose values in the children with diabetes, suggesting that the increased PG production is not related to carbohydrate homeostasis. Glycosylated hemoglobin levels have been shown to correlate with serum and urine sugar values, ~2, ~:~ and comparison of these to PG production may be of value in future studies. Because glycosylated hemoglobin concentration represents average glycemia over the previous weeks, correlation or lack of correlation with PG production might be a more reliable indicator of the possible impact of blood sugar levels. Excessive PG production may be associated with the increased platelet coagulation described in patients with diabetes' and may be related to other problems such as diabetic retinopathy or nephropathy. Prostaglandins have been associated with increased intraocular pressure, increased uveal blood vessel permeability, and with acute ocular inflammation in laboratory animals. ~4.2~ A previous report described a lower incidence of diabetic retinopathy than was anticipated in 34 patients who had both diabetes and rheumatoid arthritis. ~ This may have been related to the ingestion of aspirin for the rheumatoid arthritis and the secondarily decreased PG production. The role of PGs in the kidney is not well defined, but PGE2 increases renal blood flow and fluid, sodium, and chloride excretion in a dose-response manner in the dog kidney. ~7 It is possible that PGs could be related to either diabetic retinal or renal pathophysiology. In general, it was surprising that greater alterations in the fatty acids, and particularly of arachidonic acid, were not detected in the children with diabetes, Adults with diabetes who had higher fasting blood sugar values have been shown to have lower quantitative levels of arachidonic acid than patients with lower blood sugar levels. '~ Adult patients with complications had a lower percentage of arachidonic acid than did those without complications. ~-~ In laboratory animals, the activity of the 6desaturase enzyme (Figure), which is rate limiting in the conversion of linoleic acid to arachidonic acid, is reduced by increased blood sugar concentration, '-'8.'-'~'by a lack of insulin,_~.... and by increased glucagon, epinephrine, or cyclic AMP. ~' All of these may be altered in juvenile diabetes. If the 6-desaturase enzyme were similarly influenced in children with diabetes, reduced arachidonic acid levels would have been expected to correlate with poor glucose control, As some of the children in this study were in poor control and their arachidonic acid levels were not reduced, it would seem that either the animal data do not pertain to children with diabetes, or that a more severe degree of poor control is necessary to produce alteration of arachidonic acid levels. The present study
Volume 94 Number 2
does not suggest that determination of individual fatty acid levels in plasma or in RBCs will add to information about glycemic control in children with diabetes mellitus. As in a previous study, = cholesterol levels were elevated in 25% of the children with diabetes in this study. Prostaglandin E1 values correlated positively with cholesterol levels at incubation times of 20 minutes and 70 minutes. The biochemical basis of the correlation between cholesterol and PGE1 levels is still obscure. Appreciation is expressed to Noreen Welch, Carol Dabiere, Jane Wilson, Mary Rabaglia and Patricia T. Connally for technical assistance, to David Goldgar for help in statistical analyses, and to Ellen Cline and Dr. Vicken Totten for help in manuscript preparation. REFERENCES
1. Silver MJ, Smith JB, Ingerman C, and Kocsis JJ: Human blood prostaglandins: formation during clotting, Prostaglandins 1:429, 1972. 2. Veale WL, Cooper KE, and Pittman QJ: Role of prostaglandins in fever and temperature regulation, in Ramwell PW editor: The prostaglandins, Vol 3, New York, 1977, Plenum Press, p 145. 3. Gorman RR, Bunting S, and Miller OV: Modulation of human platelet adenylate cyclase by prostacyclin (PGX), Prostaglandins, 13:377, 1977. 4. Halushka PV, Lurie D, and Colwell JA: Increased synthesis of prostaglandin-E-like material by platelets from patients with diabetes mellitus, N Engl J Med 297:1306, t977. 5. Egeberg O: Blood coagulability in diabetic patients, Scand J Clin Lab Invest 15:533, 1963. 6. Bridges JM, Dalby AM, Millar JHD, and Weaver JA: Effect of D-glucose on platelet stickiness, Lancet 1:75, 1965. 7. Sagel J, Colwell JA, Crook L, and Laimins M: Increased platelet aggregation in early diabetes mellitus, Ann Intern Med 82:733, 1975. 8. Bensoussan D, Levy-Toledano S, Passa P, et al: Platelet hyperaggregation and increased plasma level of von Willebrand factor in diabetics with retinopathy, Diabetologia 11:307, 1975. 9. Fleischman AI, Bierenbaum MI, Stier A, Somol H, and Watson PB: In vivo platelet function in diabetes mellitus, Thromb Res 9:467, 1976. 10. Colwell JA, Sagel J, Crook L, Chamber A, and Laimins M: Correlation of platelet aggregation, plasma factor activity and megathrombocytes in diabetic subjects with and without vascular disease, Metabolism 26:279, 1977. 11. Schrade W, Boehle E, Biegler R, and Harmuth E: Fattyacid composition of lipid fractions in diabetic serum, Lancet 1:285, 1963. 12. Tuna N, Frankhauser S, and Goetz FC: Total serum fatty acids in diabetes: relative and absolute concentrations of individual fatty acids, Am J Med Sci 255:120, 1968. 13. McCosh EJ, Meyer OL, and Dupont J: Radioimmunoassay of prostaglandins El, E~, and F2a in unextracted plasma, serum and myocardium, Prostaglandins 12:471, 1976.
Prostaglandin synthesis in diabetes mellitus
189
14. Nelson GJ: Extraction of plasma and serum, in Nelson GJ, editor Blood lipids and lipoproteins: Quantitation, composition, and metabolism, New York, 1972, John Wiley & Sons, Inc., p 10. 15. Rouser G, Kritchevsky G, and Yamamoto A: In Marinetti GV, editor: Lipid chromatographic analysis, New York, 1967, Marcel Dekker, Inc., pp 99-162. 16. O'Brien D, Ibbott FA, Rodgerson DO: Laboratory manual of pediatric microbiochemical techniques, ed 4, New York, 1968, Harper & Row, Publishers, Inc., pp 153-t56. 17. Chase HP, O'Quin RJ, and O'Brien D: Screening for hyperlipidemia in childhood, JAMA 230:1535, 1974. 18. Hwang DH, Mathias MM, Dupont J, and Meyer DL: Linoleate enrichment of diet and prostaglandin metabolism in rats, J Nutr 105:995, 1975. 19. Dupont J, Mathias MM, and Connally P: Prostaglandin synthesis as a function of dietary linoleate concentration, Fed Proc 37:445, 1978. 20. Pace-Asciak CR: Oxidative biotransformations of arachidonic acid, Prostaglandins 13:811, 1977. 21. Lands WEM, and Rome LH: Inhibition of prostaglandin biosynthesis, in Karim SMM, editor: Prostaglandins: Chemical and biochemical aspects, Baltimore, 1976, University Park Press, p 87. 22. Gonen B, Rubenstein AH, Rochman H, Tanega SP, and Horwitz DL: Haemoglobin AI: An indicator of the metabolic control of diabetic patients, Lancet 2:734, 1977. 23. Gabbay KH, Hasty K, Breslow JL, et al: Glycosylated hemoglobins and long term blood glucose control in diabetes mellitus, J Clin Endocrinol Metab 44:859, 1977. 24. Whitelocke RAF, and Eakins KE: Vascular changes in the anterior uvea of the rabbit produced by prostaglandins, Arch Ophthalmol 89:495, 1973. 25. Neufeld AH, and Sears ML: Prostaglandin and eye, Prostaglandins 4:158, 1973. 26. Powell ED, and Fietcl RA: Diabetic retinopathy and rheumatoid arthritis, Lance/2:17, 1964. 27. Fulgraff G, Brandenbusch G, and Heintze K: Dose response relation of the renal effects of PGE1, PGE2, and PGF2a in dogs, Prostaglandins 8:21, 1974. 28. de Gomez Dumm INT, de Alaniz MJT, and Brenner RR: Effect of diet on linoleic acid desaturation and on some enzymes of carbohydrate metabolism, J Lipid Res 11:96, 1970. 29. Peluffo RO, de Gomez Durum INT, de Alaniz MJT, and Brenner RR: Effect of protein and insulin on linoleic acid desaturation of normal and diabetic rats, J Nutr 101:1075, 1971. 30. Mercuri O, Peluffo RO, and Brenner RR: Depression of microsomal desaturation of linoleic to "~-linolenic acid in the alloxan-diabetic rat, Biochim Biophys Acta 116:409, 1966. 31. de Gomez Dumm INT, de Alaniz MJT, and Brenner, RR: Comparative effect of glucagon, dibutyryl cyclic AMP, and epinephrine on the desaturation and elongation of linoleic acid by rat liver microsomes, Lipids 11:833, 1976. 32. Chase HP, and Glasgow AM: Juvenile diabetes mellitus and serum lipids and lipoprotein levels, Am J Dis Child 130:1113, 1976.
185
Increased prostaglandin synthesis in childhood diabetes mellitus Prostaglandins are synthesized from the fatty acids, linoleic and arachidonic acids, and are associated with increased platelet aggregation as has been found in blood from patients with diabetes mellitus. In the present study blood was obtained from 40 children with diabetes and from 20 control children for measurements of fatty acid and PGE1, PGE.,_, and PGF.,a levels. The production of PGE., and PGF~a was significantly elevated in blood from the children with diabetes at all times measured. The mean quantitative plasma linoleic acid levels were also higher in the patients. The increased PG synthesis may be related to the vascular problems that occur in patients with diabetes.
H. Peter Chase, M.D,* Ricky L. Williams, M.D., Jaequeline Dupont, Ph.D.,
D e n v e r , Colo.,
and
F o r t Collins, Colo.
PROSTAGLANDINS are synthesized in almost all body tissues, including in platelets during blood clotting? Inflammation ~-and enhancement of platelet coagulation :~ are among the many physiologic effects of PGs, and both may be important in the vascular complications of diabetes. In vitro platelet aggregation time has been shown to be shorter in persons with diabetes than in normal persons? -1~ This effect may be secondary to increased PG production. In a recent report, increased production of a "PGE-like" material during platelet aggregation in adults with diabetes compared to controls was described? Platelet aggregation time in adults with diabetes is increased toward normal following the ingestion of aspirin, 7 a known inhibitor of PG synthesis. Fatty acids, including linoleic acid which is a substrate for PG synthesis (Figure), have been described to be altered in quantity in adults with diabetes. H. 12 In the present study, the net synthesis of PGE1, PGE2, and PGF~a was measured in whole blood from control children and From the University of Colorado Medical Center, Department of Pediatrics, and Colorado State University, Department of Food and Nutrition. Supported by Public Health Service Clinical Research Center Grant RR-69, by a gift from the American Diabetes Association, Fort Collins Unit, and by a USDA, C S R S Special Project Grant. *Reprint address: Universityof Colorado Medical Center, Department of Pediatrics, 4200 E. Ninth A re., Denver, CO 80262.
0022-3476/79/200185 +05500.50/0 9 1979 The C. V. Mosby Co.
children with diabetes. All Possible regulators of PG synthesis are present in whole blood, and the use of whole blood in production of PGs during clotting is considered to represent a physiologic biopsy technique. The levels of plasma and red blood cell FAs were also measured to determine their contribution or relation to PG production.
METHODS Forty children with insulin-dependent diabetes mellitus and 20 control children had blood drawn from the antecubital vein following an 1i- to 12-hour fast. All control and diabetic children were between the ages of 3 and 19 years, with both groups having similar ag e and sex distributions. Informed consent was obtained from the parents of all test and control children. All o f the test children were known to have had diabetes for at least one year and were otherwise well at the time of testing. Morning insulin injections were not given until after venipuncture. Prostaglandins. Net synthesis of PGE1, PGE,, and PGF2a by whole blood during clotting at 37~ was determined 10, 20, and 70 minutes following venipuncture. Synthesis was stopped by removal of blood cells by centrifugation, and the resulting serum was frozen for later radioimmunoassay?3 Sera from control and diabetic children were assayed simultaneously, with the assayer (J.D.) unaware of which group the samples were from. Fatty acids. Fatty acids were determined by a modifi-
Vol. 94, No. 2, pp. 185-189
1 86
Chase, Williams, and Dupont
The Journal of Pediatrics Febrtr 1979
"Desaturation Pathway" ~linolenic acid (18:3A6,9,12)
to other prostaglandins includlng PGa 2 and PGF2a
to: PGE 1 and PGFIa
6-de saturase /
LinoleLc Acid (18:2~9,12)
Elongation enzyme
T
.T/
Dihomogmmalinoleic
-.....
/
acid
~_ A r a c h i d o n i c Acid (20:4A5,8,11,14)
(20:3A8,11,14)
Eicosadlenoic Acid
(20:2611,14)
"~"'"~Eicosatrienoic Acid (dead end) (20:3A5,11,14)
"Elongation Pathway" (minor)
Figure. Metabolism of linoleic acid to arachidonic acid and prostaglandins. Other products of arachidonic acid, including endoperoxides and newly diagnosed PGs, have been reviewed recently.-'" cation of a previously described method." Plasma was separated from the RBCs, which were then washed three times with 0.9% saline. The FAs were extracted from the plasma and the RBCs with 2:1 chloroform:methanol containing 0.005% butylated hydroxytoluene as antioxidant. The RBC extracts were heated in a 60~ water bath for one hour and then both the plasma and RBC extracts were shaken mechanically for 30 minutes to obtain maximal extraction of FAs. Samples were methylated by heating at 60 ~ for 30 minutes with boron trifluoride in methanol. Fatty acid methyl esters were then extracted with petroleum ether, concentrated, and measured using a Hewlett-Packard gas chromatograph with automatic integrator computer. A coiled glass column, 180 cm x 2 mm, packed with I0% SP 2340 on 100/120 mesh Chromosorb W AW (Supelco) was used. Identification was made by comparison to pure standards and quantification was achieved by recovery of a C15 internal Standard~ Total FAs were calculated as the sum of individual values. All extractions were completed within 48 hours of venipuncture. Phospholipids were purified following the initial chloroform:methanol extraction (above) using Unisil column chromatography. '5 Preliminary experiments demonstrated that 99% of radioactivity was recovered following Unisil column chromatography. After column purification, the samples were methylated and analyzed on the gas chromatograph as above. Measures of diabetic control. All children with diabetes were asked to bring in a 24-hour urine specimen at the time of blood testing to measure glucose spillage as an index of glycemic control. Creatinine measurements to verify that the sample was a 24-hour collection were not made, but in most cases the children had parental supervision during the collection, and most were already
familiar with the technique. Urine and serum glucose levels were measured by the glucose oxidase method. ''~ Blood was obtained from the children with diabetes for fasting serum glucose levels and for cholesterol and triglyceride levels, as previously described, '7 when the samples were drawn for PG and FA analyses. Statistical analyses. Differences between the patient and control groups were tested using the Student t test, for which P values are given. Correlation coefficients for the values of children with diabetes were computed using the Pearson formula, and r values are included. RESULTS
Prostaglandin levels. Serum PGE1, PGE~, and PGF~a values for 20 control children and 40 children with diabetes are shown in the Table. Prostaglandin E., and PGF~a levels were significantly greater in diabetic children compared to controls at all three incubation times measured, i.e., 10, 20, and 70 minutes. Prostaglandin E1 values were significantly higher in the test children only at 20 minutes. The most frequently elevated PGs (above 2 SD of normal at any of the three times studied) in the children with diabetes were PGE~ (17 children) and PGFz a (19 children). Ten children had elevation of PGE, at any of the three times. Some children who had elevated PG levels at one incubation time did not have elevated levels at a different incubation time. The number of children with elevations at each incubation time is shown in parentheses in the Table. Three children had elevations ( > 2 SD of normal) of all 3 PGs (at one or more of the three time periods), whereas four children did not have elevations of any PG at any time measured. Including the three children who had elevations of all three PGs, five had elevations (at any of
Volume 94 Number 2
the times measured ) of both PGE, and PGE~, i l had elevations of PGE~ and PGF2a, and six had elevations of PGE1 and PGF2a. Prostaglandin levels did not significantly (P > 0.05) correlate with age, sex, or duration of diabetes in the diabetic children. Further analyses were done using the Student t test, in which the children with diabetes were divided into those with: (1) an elevated level of one of the three PGs (at any of the three time periods), or (2) a normal level (at all time periods) of the same PG. There were again no significant (P > 0.05) differences between the two groups for age, sex, or duration of diabetes for any of the three PGs. Linoleic acid levels were significantly higher (P < 0.05) in the group with elevated PGE~ values, and cholesterol levels were higher in those with elevated PGE1 (P < 0.01), compared to the values for diabetic children with normal levels of the corresponding PG. Other comparisons between the two groups were not significantly different (P > 0.05). Plasma fatty acids. The percentages of individual FAs in plasma from the diabetic and the control children were similar except for stearic acid (18:0), which was higher (P < 0.01) in the 20 control children (11.5 _ 2.3%; mean ___1 SD) than in the 40 diabetic children (9.9 _ 1.4%). When FAs were expressed quantitatively, in mg/dl, palmitic acid (16:0) was significantly higher (P < 0.05) in the children with diabetes ( 4 7 . 1 _ 10.4) than in the control children (42.6 _+ 6.6), as were oleic acid (18: 1A9) (diabetic children: 45.2 _ 15.5 vs controls: 36.5 -4- 7.3; P < 0.01) and linoleic acid (18:2A9,12) (diabetic children: 60.0 _+ 17.2 vs controls: 51.5 +__ 13.5; P < 0.05). Total FAs, expressed in mg/dl, were also higher (P < 0.01) in the 40 children with diabetes (200.2 ___ 45.2) as compared to the 20 control children (174.8 _ 27.0). Plasma phospholipid FAs were determined on the first 15 patients and the first 11 control children, and as there were no significant differences (P > 0.05) between the two groups of children, no further analyses were done. RBC fatty acids. The percentage and quantitative levels of RBC FAs were similar for the control children and the children with diabetes for all FAs except palmitoleic acid (16:1A7) (test children: 0.9 _+ 0.5% vs control children: 1.9 _+ 1.5%; P < 0.01). Since it was apparent after analysis of RBC FAs on the first 15 children with diabetes that values were very similar to those of control children, further samples for RBC FAs were not obtained. There were no significant differences (P > 0.05) in RBC phospholipids between the first 11 control and the first 15 test children.
Prostaglandin: fatty acid correlations for the children with diabetes. Prostaglandin E~ levels (at all three times measured) correlated positively with mg of plasma linole-
Prostaglandin synthesis in diabetes mellitus
18 7
Table. Prostaglandin production in the blood of control children and of children with diabetes*
Incubation time
PGE1 Control children Diabetic children
PGE., Control children Diabetic children
PGF~a Control children Diabetic children
1Omen
[ 20rain
] 70 min
1.22 -+ 1.36 2.82 +4.41 (5)
2.72 _+2.47 6.17 -+6.65t (9)
7.01 -+3.17 9.65 +8.22 (5)
0.33 --+0.74 1.56 _ 1.941" (6)
1.31 -4-1.60 4.22 -- 3.975 (9)
2.49 ' -+ 1.16 5.92 -+4.00w (15)
0.20 -- 0.77 1.08 _+1.70t (6)
0.81 -----1.50 3.76 --+3.90w (9)
1.85 - 1.21 6.96 _+4.73w (19)
*Results are expressed in ng/ml and represent the mean + 1 SD for 20 control children and 40 diabetic children. The number of children with elevations (>2 SD of normal) at each of the time periods is shown in parentheses. tP < 0.05. ~P < 0.01. w < 0.001. ic acid (P < 0.05, r = 0.36), and correlated with mg of oleic acid (P < 0.05, r = 0.35) and total fatty acids (P < 0.05, r = 0.35) at 70 minutes. Prostaglandin E~ levels correlated with mg of plasma linoleic acid (P < 0.05, r = 0.37), oleic acid (P < 0.05, r = 0.36) and total FAs (P < 0.05, r = 0.34) at 70 minutes. No other FAs correlated significantly with PGE, or PGE~ levels, and PGF~ a levels did not correlate with levels of any FA.
Measures of diabetic control (glucose, triglyceride, cholesterol). Serum glucose levels were < 150 m g / d l in 11 of the patients, from 151 to 299 m g / d l in 23 children, and > 3 0 0 mg/dl in six children. Serum glucose levels did not correlate significantly with specific FA or P G values, or with cholesterol and triglyceride levels. Urine glucose levels were < 5 0 gm/24 hours in 24 patients, between 50 and 100 gm/24 hours in eight patients, and > 100 gm/24 hours in eight children. Urine glucose spillage correlated with serum glucose levels at P < 0.001 (r = 0.49). Total FA levels in patients who spilled > 5 0 gm glucose were 215.4 mg/dl, compared to 190.1 mg/dl (P = 0.08) in those spilling < 5 0 gm. Urine glucose spillage did not correlate significantly with levels of specific FAs or PGs.
18 8
Chase, Williams, and Dupont
Serum triglyceride values were normal ( < 130 mg/dl) 17 in 22 children, between 130 and 200 mg/dl in 10, and >200 mg/dl in eight children. Total FA values were 213.3 mg/dl in children with triglyceride levels > 130 mg/dl, Compared to 190.6 mg/dl in those with levels < 130 mg/dl (P = 0.11). The percentages of palmitic and stearic acids, but not of other FAs, were significantly higher (P < 0.05) for children with triglyceride levels > 130 mg/dl than in children with values < 130 mg/dl. Triglyceride values did not correlate significantly with specific PG levels. Serum cholesterol levels were in the normal range (<210 mg/dl)" in 30 children; 10 children had values that were >210 mg/dl. Six of the 10 children had both elevated serum cholesterol and triglyceride values. Cholesterol values correlated with levels of palmitic (P < 0.05, r = 0.29), palmitoleic (P < 0.02, r = 0.35), stearic (P < 0.002, r = 0.44), linoleic (P < 0.02, r = 0.36) and dihomogammalinolenic (20:32x5,8,11) ( P < 0 . 0 5 , r = 0.27) acids. Cholesterol levels also correlated with PGE, levels at 20 minutes (P < 0.005, r = 0.52) and at 70 minutes (P < 0.05, r = 0.37) incubation. DISCUSSION Mean PGE2 and PGF2a production was significantly elevated in blood from children with diabetes at all times studied (Table). Linoieic acid is currently believed to be important in regulating PG synthesis and was significantly higher in the Plasma of Children with diabetes compared to control children. Linoleic acid is the dietary source of dihomogammalinolenic acid and arachidonic acid, which are the substrates for PG synthesis (Figure). A deficiency of linoleic acid in the diet has been shown to result in decreased PG synthesis, whereas an excess of dietary linoleic acid caused increased PG synthesis during blood dotting.~8, a9 In the present study, linoleic acid levels correlated significantly with PGE~ values at all three times measured, and with PGE2 values at 70 minutes in the children with diabetes. However, it is likely that other factors were also involved in the elevated PG production. All three PGs should have been similarly elevated if Substrate alone was the cause of increased PG synthesis, but elevations of the two-series of PGs were found more frequently than of the one-series. Current knowledge of PG metabolism indicates that ratios of various PG and thromboxane compounds are important. ~~ Regulation of the synthesis of PGs involves oxygen, glutathione, peroxidases, vitamin E, selenium, and levels of fatty acids. '~1 Future studies should be aimed at determining whether manipulation of the ratios, inhibition of synthesis, or altering other factors regulating PG synthesis might have an effect on decreasing the vascular complications of diabetes.
The Journal of Pediatrics February 1979
There were no statistically significant correlations between PG levels and urine or serum glucose values in the children with diabetes, suggesting that the increased PG production is not related to carbohydrate homeostasis. Glycosylated hemoglobin levels have been shown to correlate with serum and urine sugar values, ~2, ~:~ and comparison of these to PG production may be of value in future studies. Because glycosylated hemoglobin concentration represents average glycemia over the previous weeks, correlation or lack of correlation with PG production might be a more reliable indicator of the possible impact of blood sugar levels. Excessive PG production may be associated with the increased platelet coagulation described in patients with diabetes' and may be related to other problems such as diabetic retinopathy or nephropathy. Prostaglandins have been associated with increased intraocular pressure, increased uveal blood vessel permeability, and with acute ocular inflammation in laboratory animals. ~4.2~ A previous report described a lower incidence of diabetic retinopathy than was anticipated in 34 patients who had both diabetes and rheumatoid arthritis. ~ This may have been related to the ingestion of aspirin for the rheumatoid arthritis and the secondarily decreased PG production. The role of PGs in the kidney is not well defined, but PGE2 increases renal blood flow and fluid, sodium, and chloride excretion in a dose-response manner in the dog kidney. ~7 It is possible that PGs could be related to either diabetic retinal or renal pathophysiology. In general, it was surprising that greater alterations in the fatty acids, and particularly of arachidonic acid, were not detected in the children with diabetes, Adults with diabetes who had higher fasting blood sugar values have been shown to have lower quantitative levels of arachidonic acid than patients with lower blood sugar levels. '~ Adult patients with complications had a lower percentage of arachidonic acid than did those without complications. ~-~ In laboratory animals, the activity of the 6desaturase enzyme (Figure), which is rate limiting in the conversion of linoleic acid to arachidonic acid, is reduced by increased blood sugar concentration, '-'8.'-'~'by a lack of insulin,_~.... and by increased glucagon, epinephrine, or cyclic AMP. ~' All of these may be altered in juvenile diabetes. If the 6-desaturase enzyme were similarly influenced in children with diabetes, reduced arachidonic acid levels would have been expected to correlate with poor glucose control, As some of the children in this study were in poor control and their arachidonic acid levels were not reduced, it would seem that either the animal data do not pertain to children with diabetes, or that a more severe degree of poor control is necessary to produce alteration of arachidonic acid levels. The present study
Volume 94 Number 2
does not suggest that determination of individual fatty acid levels in plasma or in RBCs will add to information about glycemic control in children with diabetes mellitus. As in a previous study, = cholesterol levels were elevated in 25% of the children with diabetes in this study. Prostaglandin E1 values correlated positively with cholesterol levels at incubation times of 20 minutes and 70 minutes. The biochemical basis of the correlation between cholesterol and PGE1 levels is still obscure. Appreciation is expressed to Noreen Welch, Carol Dabiere, Jane Wilson, Mary Rabaglia and Patricia T. Connally for technical assistance, to David Goldgar for help in statistical analyses, and to Ellen Cline and Dr. Vicken Totten for help in manuscript preparation. REFERENCES
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