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ESTIMATION OF HYDROGEN PEROXIDE LEVELS IN THE BLOOD AND URINE OF NORMAL INFANTS AND INFANTS WITH SEPSIS
A V Pherwani, V C Puri, V Malhotra
Dept. of Paediatrics and the Dept of RIA, PD Hinduja National Hospital and Medical Research Centre
We estimated H2O2 levels in normal infants’ blood and urine (NIB and NIU) and compared them with values in blood and urine (SIB and SIU) of infants with sepsis.
Method : H2O2 was measured by radioisotopic method by measuring the 14-CO2 liberated after it had decarboxylated 1-14-alpha ketoglutaric acid.
Results : Mean values for NIB (5.75, SD = 2.5 uMol/L) and NIU (17.4, SD = 10.18 uMol/L) have been estimated for the first time in India. No NIB values are available in the literature. Our NIU values are much lower than those reported for adults. [2, 4] Though H2O2 was elevated in both SIU (mean = 74, SD = 5.33) and SIB (mean = 35.7, SD = 23.6), the levels were showed a greater rise in SIU than SIB (p= <) indicating that urine is a better parameter than the blood for estimating H2O2levels.
Conclusions : It is possible to measure accurately H2O2 levels in the blood and urine of infants. Values for NIB and NIU have been estimated for the first time. Values in the urine were higher than in the blood in normal infants as well as in infants with sepsis, indicating that the urine is a better indicator of sepsis. Our NIU values are much lower than those reported for adults. [2, 4] Taking H2O2 levels in normal cases as a basis, at least 400 sample test results of normal and septic cord blood are needed to confirm these data. Estimating H2O2 levels can be helpful in diagnoses and follow up of sepsis.INTRODUCTION
During its normal metabolism in the body, 5% of the oxygen undergoes monovalent reduction (Redox cycle), generating a number of intermediate metabolites and oxygen radicals (ROS). ROS have a physiological role to play in the body. Excessive production of ROS (infections, ischaemia, reperfusion, labour, prematurity, radiation drugs and oxygen therapy) which overwhelm the anti oxidant systems[1] can result in disease. H2O2 is also an active O2 but not an oxygen radical as it does not have an unpaired electron. Amongst O2 radicals, measurement of H2O2 is comparatively easier as it has a long half life. At present only a few in vitro studies on the ability of polymorphs to produce H2O2 have been reported. Only a few studies have been reported in adult human urine, cornea, cord blood and breath.
The aim of the study was to estimate and compare H2O2 levels in urine and blood of normal infants, estimate urine and blood H2O2 levels in infants with sepsis and to determine if urine estimations can be used alone as an indicator of sepsis thus avoiding repeated blood sampling.METHODS
The data sample comprised urine and blood samples of normal infants and those with sepsis (1-12 months). Diagnosis of sepsis was made clinically according to the established criteria[2] and confirmed by blood culture. Samples were not included if the blood culture was subsequently found to be negative. Serum was immediately separated from all blood samples. All samples were stored in a frozen state. A written permission was obtained from the parents. Student’s test for paired samples and Wilcoxon’s rank order test for unpaired data were utilised for statistical analysis.MEASUREMENT OF H2O2 (RADIOISOTOPIC METHOD)
The method is based on Mathru’s method[3] with modifications. Decarboxylation of I-14 C-alpha-ketoglutaric acid* was carried out by reaction with H2O2 counter. The reaction was carried out in a test tube (8.5 x 1.5 cm) with a side arm (2 x 0.5 cm). The tube was custom made by us. A mixture of radiolabelled* and non-labelled** alpha-ketoglutaric acid was placed in the test tube*** which was covered with a CO2 trap, which is a tube with holes containing soda lime, kept on top of the reaction tube. The samples were diluted 1:20 with tyrode buffer and injected through the rubber stoppered side arm. After incubation period, the sample was acidified with trichloroacetic acid and incubated further. The trap was then transferred to a scintillation vial. The liberated[14] CO2 is counted in a liquid scintillation counter. The samples were subject to internal quenching and the results computed directly from the DPM calculated, considering the efficiency of the beta spectrometer for C14. The counting was done in the beta counter.
DPM (sample) - DPM
H2O2 (umol/L) =(background) x dilution factor / DPM (reference)
DPM = disintegrations per minute
*New England Nuclear Co., Sico Bhavan, Mackwana Rd. Andheri, Mumbai - 400 069.
**Sigma-Aldrich Corp., PO Box 20, Himayat Nagar, Hyderabad - 500 029, AP.RESULTS
As shown in Table the data sample comprised normal infant blood (NIB, n=23), normal infant urine (NIU, n=21), blood of infants with sepsis (SIB, n=18) and urine of infants with sepsis (SIU, n=23). The maximum NIB levels were 8.2 and the minimum 1.07 (mean = 5.75, SD = 2.53) and the maximum NIU levels were 21 and minimum 5.9 (mean = 17.4, SD 10.18) uMol/L. The high standard deviation could probably be due to the small sample size. There are no reported values available for NIB. The values by Mathru et al[2] are 88 + 4 and by Varma et al[4] are 100+ 60 uMol/L for adult urine. The levels were significantly higher (P = < 0.01) in the NIU than NIB. The SIB levels (maximum = 66, min.=4.78, mean = 35.71, SD = 23.56) were significantly lower (p= < 0.0) than the SIU (maximum = 136, minimum = 48, mean = 74, SD = 5.33) uMol/L. We collected matched blood (MNIB) and urine (MNIU) samples of 6 normal infants at the same time to compare these levels. MNIB (mean = 4.41,SD = 0.83 uMol/L) were significantly lower (p = < 0.05) than MNIU (mean = 26.85, SD = 9.46 uMol/L).
NIB
NIU
SIB
SIU
1.07 16.70
23.56
136.00
3.40
17.20
40.56
57.85
3.17
22.80
62.00
55.20
1.64
9.64
40.00
58.00
3.10
10.45
48.00
54.00
1.33
36.20
68.00
62.70
2.70
13.66
58.00
58.90
5.52
51.50
66.00
59.94
5.52
16.87
46.00
79.80
7.20
18.01
48.00
136.00
8.12
15.40
62.00
58.00
6.52
16.20
40.00
96.00
7.74
15.70
7.35
97.74
8.10
20.10
4.78
89.10
6.50
12.96
9.81
76.50
7.70
5.90
5.41
77.70
5.60
7.80
5.20
65.60
8.20
6.70
9.19
88.20
8.20
18.60
68.90
2.70
15.32
62.70
5.60
12.30
55.60
8.20
48.20
8.20
59.80
Mean
5.75
17.14
35.71
74.00
SD
2.53
10.18
23.56
5.33
n
23.00
21.00
18.00
23.00
MAX
8.20
51.50
66.0
136.00
MIN
1.07
5.90
4.78
48.20
NIB = normal infant blood,
NIU = normal urine,
SIB = blood of infant with sepsis,
SIU = urine of infant with sepsisDISCUSSION
Normal NIB were between 2.53 and 5.75 and NIU were between 10.18 and 17.4 uMol/L. To confirm that the levels in the urine are higher than in the blood we studied matched blood (MNIB) and urine (MNIU) samples in individual normal infants (n=5). MNIU was significantly higher than MNIB (p = < 0.05). There are no reported NIB and NIU values in infants.
Our NIU values are much lower than those for adults. [2, 4] Both, the NIB and NIU are much lower than SIB and SIU. Values in the urine were higher than in the blood in normal infants as well as in infants with sepsis, indicating that the urine is a better indicator of sepsis, avoiding the need for repeated blood sampling. Taking H2O2levels in normal cases as a basis, at least 400 sample test results of normal and septic cord blood are needed to confirm these data. Estimating H2O2 levels can be helpful in diagnoses and follow up of sepsis.CONCLUSIONS
It is possible to measure accurately H2O2 levels in the blood and urine of infants. Values for NIB and NIU have been estimated for the first time. Values in the urine were higher than in the blood in normal infants as well as in infants with sepsis, indicating that the urine is a better indicator of sepsis. Our NIU values are much lower than those reported for adults. [2, 4] Taking H2O2 levels in normal cases as a basis, at least 400 sample test results of normal and septic cord blood are needed to confirm these data. Estimating H2O2 levels can be helpful in diagnoses and follow up of sepsis.ACKNOWLEDGEMENTS
We are thankful to The Research society, The PD Hinduja National Hospital and Medical Research Centre for providing funds for the project.
We are thankful to The department of Paediatrics, LTMG Hospital and LT Medical College for their help in providing blood and urine samples.REFERENCES
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