Plasma 6-keto-PGF1α, thromboxane B2 and PGE2 during diabetic ketoacidosis

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Leukotrieaes and Eaential Fatty Acids (1990)40.39-43

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Plasma 6-keto-PGF lar, Thromboxane B2 and PGE2 during Diabetic Ketoacidosis T. Mourits-Andersen,

I. W. Jensen, L. K. Nielsen, G. L. Nielsen, J. Dyerberg and J. Ditzel

Department of Medicine, Section of Endocrinology and Metabolism and Department of Clinical Chemistry, Aalborg Hospital, Aalborg, Denmark (Reprint request to TMA) ABSTRACT.

In 10 patients admitted to hospital with diabetic ketoacidosis plasma prostanoids Cketo-PGF a, thromboxane Bz and PGQ were studied hefore treatment and following recovery. buring ketoacidasis the median plasma 6-keto-PGFI, and PGE2 were significantly increased compared to those of a normal reference group: 5.2 pg/ml and 3.9 pg/mI versus 1.7 pg/mI and 0.4 pg/ml (p< O.Oland p
thromboxane B2 and PGE;? between Type 1 diabetic patients and healthy subjects. During exercise the plasma 6-keto-PGF1ar increased significantly but to the same degree in the diabetic patients and the healthy subjects and thus, demonstrated no indication of a decreased capacity of prostacyclin production in Type 1 diabetic patients (16). For further elucidation of the role of prostanoids in various diabetic situations we have studied plasma 6-keto-PGF,,, thromboxane Bz and PGE2 during diabetic ketoacidosis.

INTRODUCTION Based on in vitro studies it has been suggested that insulin-dependent diabetic patients exhibit a decreased vascular capacity to produce the vasodilatory and antiaggregatory prostacyclin (PG12) and an increased platelet derived production of the vasoconstrictor and proaggregatory thromboxane A2 (TXA2). It has been proposed that these changes might be essential for the development of microvascular complications (l-5). However, whether these changes actually take place in vivo is still unsettled. Measurements of prostacyclin and thromboxane A2 production assessed from the concentrations of the stable metabolites in plasma, 6-keto-PGF1cu and of thromboxane B2 have given conflicting results which, at least partly, appear to be explained by methodological problems (6-14). By refining the method by performing high pressure liquid chromatography (HPLC) prior to the radioimmunoassay (RIA) , we found no differences either in the basal levels (15) or during standardised exercise (16) of plasma 6-keto-PGF,,,

SUBJECTS AND METHODS During 12 months, 10 patients (2 males and 8 females) admitted to Aalborg hospital with diabetic ketoacidosis were investigated. The diagnosis of diabetic ketoacidosis (DKA) was based on excessive ketonuria, a plasma bicarbonat ~18 mmol/l and /or arterialblood pH<7.3. Nine of the patients had previous to admission been treated with insulin but one (case 4) was in treatment with Glibenclamide and Metformin. The median age was: 20 years, blood glucose: 27.0 mmol/l and HbAI,: 11.1 %. None of the patients showed clinical evidence of macrovascular complications or had signs of diabetic

Date received 20 September 1989 Date accepted 20 November 1989 39

40 Prostaglandins Leukotrienes and Essential Fatty Acids

nephropathy. Only one patient had simple retinopathy. The diabetic patients served as their own controls. However values were compared with those of a healthy reference group consisting of 25 volunteers (13 males, 12 females) with a median age: 31 years, blood glucose: 5.0 mmol/l9 HbA1,: 4.9 %. None were receiving medication other than insulin nor had any taken non-steroidal anti-inflammatory drugs (NSAID) within the previous week. None were alcohol-dependent, nor had they ingested alcohol during the 48 h prior to the examination. None were allowed to smoke during the 2 h before the examination. Informed consent was obtained from all subjects and the study ,was accepted by the local ethical committee. Chmcal details of the 10 patients admitted in diabetic ketoacidosis are given in table 1. Immediately following admission venous blood samples for prostanoid analysis and serumelectrolyte determination were drawn in the supine position and in a non-fasting condition. All patients were treated with a conventional ‘low dose insulin’ schedule with 12 units of crystalline insulin intravenously per hour. The median duration of this regime was 29.5 h (range 22-36). In 1 patient (case 4) supplementary infusion of bicarbonate was given. The median amounts of intravenous fluid and insulin used during treatment of DKA were 10.0 l(range 7.0-13.5) and 256 IU of insulin (range 176320). After recovery from DKA (mean 13.2 days) the standardized blood tests were repeated. The concentrations of plasma bicarbonate, arterial blood pH, blood glucose and creatinine were treated with a conventional “low dose insulin” HbAi, was determined by a microcolumn method (Biorad, Richmond, Calif, USA). Plasma 6-ketoPGF1,, thromboxane B2 and PGE2 were isolated by a reverse-phase high pressure liquid chromatography system (re-HPLC) (Waters Associates, Milford, USA) and determined by a “‘1 Radioimmunoassay kit (NEN Inter-national, Boston, USA) as previously described (15-16). In brief, venous blood samples were taken from a cubital vein with a maximum of 50 mmHg stasis. Precooled polypropylene tubes were used containing 200 pi of a mixture of dipotassium ethylenediamine tetraacetic acid (K2 EDTA) 95 mg/ml and indomethacin 1.8 gp/ml. -The blood was kept on ice until centrifugation for 10 min at 200 g and 4°C to obtain platelet free plasma, which was enriched with a known amount of tritium labelled internal standards. The prostanoids 6-keto PGFi,, thromboxane B2 and PGE2 were extracted from the platelet free plasma using a SEP-PAK Cis Cartridge, and the individual prostaglandins were separated by re-HPLC and after lyophilization determined by a lz51 radioimmunoassay kit. The RIA-results were corrected for recovery after the plasma blank value and the internal standards were

subtracted. The intra/inter series variation (CV%) of the assay procedure was 8.5/12.2; 6.4/7.8; 6.2/14.6 for 6-keto-PGFi,, TXB2 and PGE2 respectively (16). The sensitivity of the radioimmunoassay, defined as two times the standard deviation at zero binding, was 0.3, 0.3 and 0.4 pghi for 6-ketoPGF,,, TXB2 and PGE2 respectively. Statistical analysis The Wilcoxon test for paired comparisons and the Mann-Whitney test for unpaired two-sample comparisons were used. Analysis of correlation between two variables were done by Sperman’s rank correlation test. A 5% (two tailed) level of statistical significance was used.

RESULTS During diabetic ketoacidosis the plasma concentrations of 6-keto-PGFisnd PGE* were significantly elevated (Table 2) as compared to the concentrations in the normal reference group ~~0.01 and pxO.05 repectively. Following treatment of DKA the median plasma concentrations of 6-keto-PGFi, and PGE2 decreased significantly (pO.15) and TXB2 did not change significantly after treatment of DKA (p>O.20). The changes in plasma 6-keto-PGFi, were negatively correlated to the changes in plasma pH (rho: -0.7788, p = 0.0135), but did not correlate with either the amount of intravenous insulin or the amount of intravenous fluid used during the treatment of DKA (rho: < -0.1281). Neither did 6-keto-PGFi, correlate to the other prostanoids measured in this study (rho: ‘~0.1758) nor to changes in blood glucose or HbA1, (rho: cO.2992). The changes in PGE;! were positively correlated to serum-creatinine at admission (rho: 0.6976, p = 0.0368) and to the amount of intravenous fluid and insulin used during treatment (rho: 0.7500, p = 0.0126) and (rho: 0.8424, p = 0.0023), respectively. PGE2, however, did not correlate significantly to the degree of acidosis (pH) (rho: 0.5799). There were no significant correlations between the amount of intravenous fluid and amount of intravenous insulin *used during treatment (rho: 0.5732, p = 0.083).

DISCUSSION Increased levels of plasma 6-keto-PGF1, and PGE2 have previously been reported in rats with experimental DKA (17). Our results indicate that

F F F F F M

F

F

M

2 3 4 5 6 7

8

9

10

20

20

19

32 21 49 16 16 41

20

years

Age

7.8

8.3

9.5

6.4 7.3 6.0 4.4 6.8 16.0

18.0

mmoI/I

Plasma bicarbonate

BG = Biguanide

7.06

7.10

7.29

7.01 7.08 6.91 6.91 7.06 7.33

7.36

Arterial PH

19.2

13.3

28.2

30.8 15.9 23.0 30.6 41.6 25.8

58.4

mmoI/I

Blood glucose

7.43” 7.43-7.45

Without ketoacidosis Median (l-3 quartil)

The following differences

7.07 7.01-7.29

10.4b 7.1-13.7

-

96

216

143 105 130 162 132 120

112

mmoI/I

Serum creatomome

104

52

56

28 20 GC+BG” 48 56 52

40

IU

dosis

Daily insulin

0.5” <0.2-2.2

5.2 2.6-10.5

c.dketoacidosis

1.7’ 0.7-3.2

Plasma 6-keto-PGF pg/mI Diabetic Control subjects patients

160190

150/80

145195

130/100 160/100 160/90 120/70 130/80 140190

110/70

mmHg

Blood pressure

20.9 11.1-39.2

37.0

35.7

36.3

36.1 37.9 37.6 37.9 37.0 37.5

37.1

C unstable diabetes cystitis cystitis influenza cystitis otitis media unstable diabetes unstable diabetes unstable diabetes unstable diabetes

factors

Possible precipitating

0.0s” <0.02-0.86

3.9 1.3-11.8

o.4d <0.02-1.8

Plasma PGE pe/mI Control Diabetic patients subjects

Temperature

versus control. *S = P
33.9 30.3-42.9

37.0 20.1-172.6

Plasma TXB pg/mI Diabetic Control subjects patients

at admission and following recovery

11.8

11.5

11.1 13.4 9.4 9.6 10.6 10.0

14.1

%

HbA1,

versus non-ketosis;

10.4 9.3-10.8

11.1 10.0-11.8

were significant: a.b ketoacidosis

26.2” 25.3-26.6

27.0 19.2-30.8

%

mmoI/l

mmoI/l

7, 6 6.4-9.5

HbA

Blood glucose

Plasma bicarbonate

data of the 10 patients with diabetic ketoacidosis

9

10

8

3 3 4 5 5 3

8

Duration of diabetes years

With ketoacidosis Median (l-3 quartil)

Arterial PH

Table 2 Laboratory

a GC = Glibenclamid

F

Sex

1

nr

Case

Table 1 Clinical data of the 10 patients admitted/ in diabetic ketoacidosis

42

Prostaglandins Leukotrienes and Essential Fatty Acids

diabetic patients in severe ketoacidosis have significantly elevated 6-keto-PGE1, and PGE2 values in plasma and that these values decrease towards normal levels following recovery. As the changes in plasma 6-keto-PGF,,were negatively correlated to changes in pH, the acidosis per se might stimulate the prostacyclin production, whereas the degree of dehydration (estimated from S-creatinine and the replacement volume of fluid) was not found to influence the 6-keto-PGFi, level in DKA. In vitro experiments an inhibitory effect of insulin and high glucose concentration on prostacyclin production has been demonstrated (N-23). In our study changes in plasma 6-keto-PGFi, did not correlate significantly with either the amount of insulin given or the plasma glucose or HbAI, concentration. This means that the insulin and glucose regulation did not directly influence the in vivo production of 6-keto-PGFi,. These results are in accordance with our previous findings (15-16). The plasma PGE2 levels were significantly positively correlated to the amount of insulin and fluid given during the treatment as well as to the Screatinine values before treatment. This means that the severity of dehydration and relative lack of insulin may influence the PGE;! production during DKA. The positive correlation between insulin dose and the decrease of PGE2 is consistent with the inhibitory effect of insulin on PGE2 production previously reported (24-25). The positive relation between dehydration and the plasma level of PGE2 in DKA in the present study does not agree with the results in rats with experimental DKA (17). Plasma thromboxane Bz was slightly but not significantly elevated in the DKA condition and did not correlate to changes in 6-keto-PGFi,, pH and bicarbonate or to diabetes regulation based on HBAi, and actual blood glucose. The result did not indicate a marked increased platelet activation in DKA, which was expected in the view of previous in vitro studies (1, 3). The elevated prostacyclin production determined as 6-keto-PGFi, and the lack of increment of plasma thromboxane B2 during DKA substantiate the observations of decreased platelet aggregation in patients with diabetic ketoacidosis previously reported (26-27). The influence of acidosis on the prostacyclin production found in our study seems to have a parallel in the increased prostacyclin biosynthesis found in patients with severe arterial ischaemia as reported by FitzGerald (28). In our previous studies (15-16) we found no significant differences in the plasma levels of prostanoids between insulindependent diabetic patients and healthy controls either in the basal condition or during a standardized exercise stimulation. These results are supported by the findings of Alessandrini and

coworkers (29). If the prostaglandins should be involved in the pathogenesis of diabetic vascular complications as suggested from in vitro studies, a decrease in plasma 6-keto-PGFi, and an increase in plasma thromboxane B2 levels should be expected in patients with diabetic ketoacidosis. In contrast the present study indicates that the plasma levels of 6-keto-PGE, are increased whereas there are normal levels of thromboxane B2 in DKA. Thus, our results do not support the hypothesis that decreased vascular prostacyclin production is a major factor in the development of vascular sequela of diabetes mellitus.

Acknowledgements This study was supported by grants from the Northern Jutland Medical Research Fund and the Danish Medical Research Council, journal no: 12-4582, 12-5250. The technical assistance of MS U. Jacobsen is highly appreciated.

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