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Technical Briefs |
1 Department of Clinical Biochemistry, Molecular Pathology, and Clinical Analytical Chemistry,2 Department of Obstetrics and Gynecology, and3 Neonatal Intensive Care Unit, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary;4 Center for Biomedical Laboratory Science, San Francisco State University, San Francisco, CA;5 Department of Laboratory, Municipal Hospital, Sümeg, Hungary
aaddress correspondence to this author at: Department of Clinical Biochemistry Molecular Pathology, Medical and Health Science Center, University of Debrecen, PO Box 70, Debrecen H-4012, Hungary; fax 36-52-417-631, e-mail goth{at}jaguar.dote.hu
Gestational diabetes occurs with variable severity in 3%–5% of all pregnancies and may be related to oxidative stress and impaired antioxidant defenses (1). Antioxidant enzymes include superoxide dismutase, which produces hydrogen peroxide, and catalase, which consumes hydrogen peroxide. Catalase is the main regulator of hydrogen peroxide metabolism (2), which is associated with diabetes mechanisms such as Glut 4 expression, insulin secretion, insulin signaling, protein tyrosine phosphatase regulation, and glucose transport stimulation (3). Hydrogen peroxide has novel insulin-like effects, e.g., inhibition of lipolysis and reactivation of phosphoenolpyruvate carboxy kinase (4)(5), and insulin moderates hydrogen peroxide generation (6)(7) and catalase synthesis (8). High concentrations of hydrogen peroxide may damage heme proteins, cause cell death, and together with redox active metal ions, produce highly toxic hydroxyl radicals.
High catalase activity in erythrocytes seems to provide antioxidant defense for tissues with low catalase activity, particularly pancreatic beta cells. Catalase is important in antioxidant defense against hydrogen peroxide (9)(10), but there are conflicting reports of decreases (11)(12), increases (13), and no change(14) in catalase activity in diabetes. A high incidence (14%) of diabetes mellitus observed in 63 Hungarian patients with inherited catalase deficiency (1 with type 1 and 7 with type 2 diabetes) could be associated with damage to oxidation-sensitive pancreatic beta cells by exposure to long-term increased hydrogen peroxide concentrations (15), but there have been conflicting reports from small studies of maternal and embryonic catalase in rat (16) and human(17) gestational diabetes.
We compared blood catalase activity in patients with gestational diabetes, pregnant patients without diabetes, and nonpregnant nondiabetic individuals. We examined the catalase gene mutations associated with decreased catalase activities and evaluated the effects of decreased maternal blood catalase activity on complications in newborns.
Study participants included 60 pregnant women with gestational diabetes but no family history of diabetes mellitus who were undergoing treatment at the Department of Obstetrics and Gynecology of the Medical and Health Science Center (Debrecen, Hungary). All received insulin therapy. Blood samples were taken from 32 women in the second trimester and 28 in the third trimester. For comparison we used 235 age- and sex-matched nonpregnant, nondiabetic women (nonpregnant group) (18) and 169 pregnant, nondiabetic women (pregnant group) from the outpatient clinics of the Municipal Hospital (Sümeg, Hungary). From this group we evaluated the catalase activity of 129 pregnant, nondiabetic women from the second trimester and 136 from the third trimester. We received consent from the participants, and the samples were deidentified.
We determined blood catalase activity with a simple spectrophotometric assay (18) [reference mean (SD), 108.6 (13.1) MU/L (n = 235) for women 20–40 years of age]. Blood hemoglobin A1c (Hb A1c) was measured with a Diamat system (Bio-Rad; (reference interval, 4.2%–6.1%), and blood hemoglobin with a blood cell counter (SF; Sysmex). Genomic DNA was extracted with a QIAamp Blood Kit (Qiagen) from leukocytes of 38 women from the nonpregnant group, 30 from the pregnant diabetic group with blood catalase activity within the reference interval, and 9 from the gestational diabetes group with blood catalase activity <50% of the mean activity in the reference population. The C-to-T regulatory mutation was examined in 22 patients with gestational diabetes with blood catalase activity below the lower limit of the reference interval (80.3 MU/L) and in the 38 nonpregnant controls. Exons 2 and 7 and the catalase promoter regions were PCR-amplified and the PCR products analyzed for known catalase gene mutations (19)(20).
We used the Student t-test to evaluate the statistical significance of differences between means. Values were considered significant at P <0.05.
Blood catalase and hemoglobin data are presented in Table 1
. The mean (SD) blood catalase activity for pregnant women [89 ( (18)) MU/L; n = 169] was decreased 18% (P <0.001) compared with the control group [109 ( (13)) MU/L; n = 235]. The decrease in mean catalase activity was even greater (32%) for the gestational diabetes group [74 ( (14)) MU/L; n = 60; P <0.001]. The mean blood hemoglobin concentrations for the pregnant nondiabetic group [120 ( (10)) g/L] and for the gestational diabetes group [117 ( (10)) g/L] were decreased 7% (P <0.001) and 9%, respectively, compared with the controls [129 ( (10)) g/L]. The mean ratio of blood catalase to hemoglobin was significantly lower (P <0.001) than in the controls [0.84 (0.10) MU/g; set as 100%] for both the nondiabetic pregnant group [0.75 (0.10) MU/g; 90% of control value] and the gestational diabetes group [0.63 (0.11) MU/g; 75% of control value].
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In the pregnant nondiabetic group, there was no difference in mean hemoglobin or catalase between the second and third trimesters, whereas in the gestational diabetes group, there was significant (P <0.05) variation of mean blood catalase [second trimester, 67 (18) MU/L (62% of control value); third trimester, 80 (15) MU/L (74% of control value)] but the mean blood hemoglobin did not vary.
We observed significant differences in the decreases in mean blood hemoglobin concentrations (6%–9%) and catalase activities (18%–38%) in the nondiabetic pregnant and gestational diabetes groups compared with the control group (P <0.05). The mechanisms that decreased hemoglobin may have decreased the catalase activity, but catalase-specific mechanisms probably caused the larger decreases in catalase activity in the women with gestational diabetes. To detect a catalase-specific mechanism, we searched for mutations in the catalase gene in samples from the women with gestational diabetes who had <50% of the normal blood catalase activity.
Mutations (19) that are known to cause catalase deficiencies in some Hungarians (exon 2, Hungarian type A and B; intron 7, type C) were not detected in any of the 38 nonpregnant individuals, 30 pregnant women without diabetes, or 9 women with gestational diabetes and catalase activity <50% of the mean activity of the reference population.
We investigated 22 patients with gestational diabetes who had decreased blood catalase activities (below the lower limit of 80.3 MU/L of the reference interval) for the catalase gene regulatory mutation (C-to-T substitution in the 5' promoter region, which may influence catalase activity (20). Although this mutation should yield the highest activities for the TT types and lowest for the CC types (20), our results showed nearly the same (P >0.7) catalase activities for the CC types [63.6 (9.7) MU/L; n = 7], CT types [62.2 (11.6) MU/L; n = 10], and TT types [64.5 (8.1) MU/L; n = 5]. Furthermore, the 38 nonpregnant individuals also showed a different trend because the highest, (nonsignificant) activity values were found for patients with the CC types [96.4 (21.1) MU/L; n = 19], lower for the CT types [93.3 (24.1) MU/L; n = 12], and lowest for the TT types [89.4 (12.8) MU/L; n = 7]. Thus, the presence or absence of these catalase gene mutations did not appear to influence catalase activity.
Before a diagnosis of gestational diabetes is made, usually during the second trimester, and before initiation of insulin therapy, oxidative stress and glucose autooxidation may increase hydrogen peroxide generation and diminish catalase synthesis (5)(6)(7)(8)(9)(10)(11)(12). The observed higher mean catalase activity in the third than the second trimester in the gestational diabetes group may reflect the success of intensive insulin therapy and decreased blood glucose and oxidative stress. This is the first report of this change in catalase activity associated with control of hyperglycemia in human gestational diabetes; our observation is consistent with animal experiments in which insulin therapy normalized catalase activity and protein expression (12).
To evaluate the change in hyperglycemia control in the gestational diabetes group, we measured Hb A1c concentrations in second and third trimester samples. Samples from both trimesters had mean Hb A1c values within the reference interval, but the second trimester mean [5.7 (0.6)%; n = 32] was significantly higher (P <0.01) than the third trimester mean [5.1 (0.8)%; n = 28]. This Hb A1c change correlated negatively with the change in mean catalase activities [second trimester, 67 (18) MU/L (n = 32) vs third trimester, 80 (15) MU/L (n = 28); P <0.001]. Similar effects have been observed for serum and blood catalase in patients with type 2 diabetes (21)(22).
The low catalase activities of the 60 insulin-treated patients with gestational diabetes were not associated with clinical disorders. During their pregnancies, 53 of these patients had no complications, 7 had hypertension, and 3 had preeclampsia.
We evaluated 53 newborns to assess the impact of decreased maternal catalase activity during gestational diabetes. The infants were divided into 2 groups based on maternal catalase activities: group A with lower [52 ( (8)) MU/L; n = 9] and group B with higher [78 ((10)) MU/L; n = 44] activity. The cutoff for groups A and B was 60 MU/L, the mean of the maternal blood catalase activities. There were no significant differences in mean birth weights [3271 (473) g for group A vs 3456 (566) g for group B] or mean gestation times [38.7 (1.2) weeks vs 38.9 (1.0) weeks]. The frequencies of cesarean section [3 of 9 (33%) vs 14 of 44 (32%)] and high (>3500 g) birth weights [4 of 9 (45%) vs 22 of 44 (50%)] were similar in these groups. Group B had a higher frequency of respiratory distress syndrome [10 of 44 (22%) vs 1 of 9 (11%)] and of other complications (icterus, 44%; hypoxia, 5%; anemia, 5%; acidosis, 5%; erythema, 5%; pyelectasy, 2%; premature delivery, 2%). Similar complications were not detected in group A, possibly because of the low number of patients in this group. Although we could not perform a clear statistical analysis because of the small number of patients, these results suggest that low maternal catalase activity does not represent a significant risk for mothers or newborns.
Our data suggest that dysregulation of catalase synthesis could be responsible for decreased blood catalase in gestational diabetes and its change in the second and third trimester. We found no link between known mutations in the catalase gene and such dysregulation, but as yet unknown mutations could be responsible for the low catalase activities associated with gestational diabetes.
Acknowledgments
This work was supported by grants from the Zsigmond Diabetes Foundation (Hungarian Academy of Science, Budapest) and the Hungarian Scientific Research Fund (OTKA TO42685).
References
-mediated insulin resistance. J Biol Chem 1999;274:25078-25084.
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