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Original Studies |
Department of Obstetrics and Gynecology (R.S., Y.Y., Y.O., S.S., K.K., J.A.), Nippon Medical School, Tokyo, 113 Japan; and the Center for Perinatal Biology (G.G.P.), Loma Linda University, Loma Linda, California 92350
Address all correspondence and requests for reprints to: Yoshio Yoneyama, M.D., Nippon Medical School, Department of Obstetrics and Gynecology, 1–1-5, Sendagi, Bunkyo-ku, Tokyo, 113 Japan.
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Abstract |
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Cordocentesis was performed in 34 growth-retarded fetuses at 31–35 weeks’ gestation for the measurement of umbilical venous plasma concentrations of hypoxanthine, hemoglobin and lactate concentrations, blood gases, and base deficit. Umbilical venous plasma hypoxanthine concentration was found to be increased significantly, in parallel with the degree of acidosis (r = -0.74, P < 0.05) and base deficit (r = -0.41, P < 0.05), but not to bear a significant relationship to the degree of hypoxemia or other measured variables. We conclude that increases in the plasma concentration of hypoxanthine may reflect an impaired physiological state in growth-retarded fetuses before labor.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The results of these previous studies suggest that the elevation of plasma hypoxanthine concentration may contribute to an evaluation of fetal asphyxia. However, other studies carried out at birth have failed to show a clear relationship between plasma hypoxanthine and the physiological state of the newborn (5, 6). The reasons for this difference are unknown but may relate to dissimilar study settings and differences in the severity of labor and fetal asphyxia.
Before labor, umbilical cord plasma hypoxanthine concentration has been evaluated only in pregnancies complicated by rhesus isoimmunization (7). In this condition, plasma hypoxanthine is not associated with other indicators of fetal asphyxia. However, because this earlier work involved study of fetuses with severe anemia, hydrops, and multiple transfusions, plasma levels of hypoxanthine may have been affected by a variety of factors in addition to hypoxia.
Previous work has demonstrated that some growth-retarded fetuses are hypoxemic and acidemic before labor (8). Therefore, it is possible that the measurement of plasma hypoxanthine concentration before the onset of labor may be a useful index of fetal asphyxia, especially in growth-retarded fetuses.
The purpose of this study was to evaluate whether the level of umbilical venous plasma hypoxanthine is correlated with the extent of fetal hypoxia and acid-base imbalance in growth-retarded fetuses before the onset of labor.
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Subjects and Methods |
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Umbilical venous blood was obtained by cordocentesis from 34 growth-retarded fetuses at 31–35 weeks’ gestation. In all cases, fetal growth was assessed by ultrasonographic measurements of fetal biparietal diameter, trunk cross-sectional area, and femur length. Growth retardation was diagnosed when estimated fetal body weight, determined by ultrasound (9), was less than the fifth percentile of the fetal growth curve of Japanese (10). The gestational age of the fetuses was established from maternal menstrual history and confirmed by ultrasonographic examination of both fetal crown-rump length at 9–11 weeks and fetal biparietal diameter at 16–18 weeks.
All mothers were healthy and nonsmokers. The indications for cordocentesis were for rapid karyotyping (n = 22) and to exclude the possibility of fetal infection (n = 12). All gravid women gave informed consent, and the study was approved by the Nippon Medical School Ethics committee.
The procedure was carried out without maternal sedation or fetal
paralysis, and no complications were noted. All infants were growth
retarded at delivery and were otherwise anatomically and chromosomally
normal. Placentas were examined macro- and microscopically by a single
pathologist, and signs of infarction and villous ischemia were taken to
indicate placental ischemia. Antenatal testing was performed by
evaluation of biophysical profile scores and umbilical artery Doppler
velocimetry before cordocentesis. A biophysical profile score of less
than 6 points was taken as abnormal. The peak systolic frequency (A)
and end-diastolic frequency (B) were measured. The pulsatility index,
calculated as (A-B)/average frequency, was determined from the mean of
five consecutive waveforms with a clear outline. A pulsatility index of
more than 2 SD above the normal mean for gestation age was
taken as abnormal. To exclude interobserver error, all Doppler
velocimetry examinations were performed by a single investigator. The
clinical description of patients is given in Table 1
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Blood samples were obtained by cordocentesis from the umbilical
vein. Samples were analyzed for plasma hypoxanthine, lactate and
hemoglobin concentrations, blood gases, pH, and base deficit. Three
samples of umbilical venous blood were taken sequentially from the free
loop of the umbilical vein through a 21-gauge needle that was guided by
ultrasonography (RT 4600, Yokogawa Medical Co, Tokyo, Japan). The first
0.2 mL of blood was collected in a heparinized syringe for measurement
of blood gases, pH and base deficit, hemoglobin concentration, and
oxygen saturation (ABL 520 and OSM-2, Radiometer, Copenhagen, Denmark).
The next 0.5 mL was collected in a heparinized syringe containing 0.1
mL of a stop solution [0.6 mmol/L erythro-9-(2-hydroxyl-3-nonyl)
adenine hydrochloride (Burroughs Welcome, London, UK)]; an adenosine
deaminase inhibitor; and 0.3 mmol/L 
, ß-methylene ADP (Sigma, St.
Louis, MO), a 5'-nucleotidase inhibitor in saline, to prevent
degradation of adenosine and inosine monophosphate to hypoxanthine
during sample collection and handling before determination of plasma
hypoxanthine concentration. A final 0.5 mL of blood was drawn into a
fluoride-oxalate tube for measurement of plasma lactate
concentration.
The samples were transferred immediately into tared tubes on ice and then centrifuged at once at 5000 x g for 5 min at 4 C. To remove plasma proteins, the supernatant was transferred to an ultrafiltration cone (Centrifree, Amicon, Beverly, MA) and deproteinized by centrifugation (1000 x g, 30 min, 25 C). The deproteinized plasma was stored at -70 C until analyzed.
Analysis of hypoxanthine and lactate levels
Plasma hypoxanthine concentration was analyzed with a modified high-performance liquid chromatographic method, as described previously (11). The mobile phase consisted of 30 mmol/L potassium phosphate and 2% methanol (vol/vol, pH 4.5), and it was degassed by vacuum extraction combined with ultrasonication for 20 min. Samples of ultrafiltrate (20 µL) were injected onto a 4.6-mm diameter x 159-mm length C18 Puresil column (Waters, Milford, MA) with a guard column (Guard-Pak, Waters) at ambient temperature. They were eluted at a flow rate of 1.0 mL/min and monitored by a variable wavelength ultraviolet monitor (655 A-21, Hitachi, Tokyo, Japan) at 254 nm. Hypoxanthine peaks were identified by retention time, coelution of standards, and enzymatic peak shifts with xanthine oxidase. The detection limit was at least 5 nmol/L, and the intra- and interassay coefficients of variation were less than 5.2% and 6.7%, respectively.
Plasma lactate concentration was assayed with an enzymatic spectrophotometric method (Diagnostic kit, Sigma) (12). The detection limit was at least 10 µmol/L, and the intra- and interassay coefficients of variation were 4.2% and 5.9%, respectively. Plasma hypoxanthine and lactate concentrations were calculated from measured concentrations after correction for dilution.
Statistical analysis
Data are presented as mean ± SEM. Statistical analyses were performed with Mann-Whitney tests and linear regression analyses using the Spearman rank test. Multivariate regression also was performed to evaluate the independent effects of measured variables on plasma hypoxanthine levels. Differences were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. Umbilical
venous plasma hypoxanthine levels averaged 2.96 ± 0.32 µmol/L
in 34 growth-retarded fetuses.
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) and base
deficit (r = -0.41, P < 0.05). Umbilical venous
plasma hypoxanthine levels did not show a significant correlation with
other measured parameters. Using multivariate regression to evaluate
the independent effects of measured variables, umbilical venous pH was
the one most significantly correlated with plasma hypoxanthine levels
(r = -0.64, P < 0.05).
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. We evaluated
the correlation between umbilical plasma hypoxanthine concentration and
fetal antenatal testing and prognosis. No significant linear
correlation was found between umbilical hypoxanthine levels and values
of umbilical artery pulsatility index, biophysical profile score, the
severity of growth retardation, delivery mode, gestational weeks at
birth, or Apgar Score. In this study, the range in gestational age of fetuses was very narrow, and measured variables did not change measurably with gestational age. It is recognized that with study of a larger number of patients and a wider range of ages, such correlations might be observed.
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Discussion |
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In the present study, growth-retarded fetuses were more hypoxemic, hypercapneic, acidotic, and hyperlacticemic before labor than we reported previously for normally grown fetuses (13). The present results are in accord with the results of a previous study of growth-retarded fetuses (8).
In the present study, carried out before the onset of labor, the mean value of plasma hypoxanthine concentration was significantly less than measured previously in nonasphyxiated fetal cord blood obtained at birth (2, 3). A number of explanations for this difference may be suggested. For example, uterine contractions may have induced fetoplacental ischemia and hypoxia and led to increased release of adenosine, conversion to hypoxanthine through the adenosine-to-uric-acid cascade, and hence, the elevated hypoxanthine levels found at birth (14). Also, the steady-state concentration of hypoxanthine is likely to differ in flowing blood exiting from a functioning placenta, as opposed to stagnant blood collected after cord clamping immediately after birth. The placenta is an active pathway for hypoxanthine elimination before birth (15). And finally, the degradation of adenosine to hypoxanthine during blood collection may have contributed to elevated hypoxanthine levels measured at birth, if this pathway were not sufficiently blocked; very low hypoxanthine levels are observed in umbilical cord blood after birth, when an adenosine deaminase inhibitor is used during sampling (14).
The present study was performed before the onset of labor and used adenosine deaminase and 5'-nucleotidase inhibitors during blood sampling. It was possible, therefore, to exclude the role played by labor stress in elevating plasma hypoxanthine and to minimize the role played by adenosine degradation during blood sampling.
In the present study, umbilical venous plasma hypoxanthine concentration was inversely related to pH and base deficit, but not to pO2 and pCO2. These results are in accord with earlier studies (3, 4). In the present study, no significant relationship was found between umbilical venous hypoxanthine and lactate levels, results which differ from earlier work showing such a correlation (2, 6). A possible explanation for this difference may relate to the type of patients enrolled for investigation. The present study was undertaken to study growth-retarded fetuses, some of which were hypoxic or acidotic but others of which were not. Lactate metabolism in growth-retarded fetuses is different from that in normally-grown fetuses (16), and elevated lactate levels have been reported during normoxia (17). Thus, lactate, hypoxanthine, and energy state need not necessarily bear the same relationship to one another in normally-grown and growth-retarded fetuses.
To date, umbilical venous hypoxanthine concentration has been reported before labor only in pregnancies complicated by rhesus isoimmunization (7). In this earlier work, no relationship was observed between umbilical venous hypoxanthine concentration and pH, and thus, there is a difference with the present results, wherein an inverse correlation was found with pH. In the earlier-studied fetuses, blood gases and lactate concentrations were within normal ranges, and alterations in hypoxanthine may have been too small to be detected. Second, earlier studied fetuses were anemic and, because adenosine concentration is closely related to hemoglobin concentration (18), adenosine (and hence, hypoxanthine) levels may have been reduced and atypical. Third, some earlier-studied fetuses were hydropic, and the changes of hypoxemia and acidemia within the fetoplacental circulation associated with hydrops (19) may have altered umbilical venous hypoxanthine concentration.
Thus, a variety of factors may have combined to obscure relationships between umbilical hypoxanthine and other variables measured in the previous study. In the present study, fetuses were not anemic, and adenosine deaminase and 5'-nucleotidase inhibitors were used to prevent the degradation of adenosine to hypoxanthine during blood sampling and handling. Perhaps because these confounding factors were minimized, it became possible to detect a correlation between umbilical venous hypoxanthine levels and pH. The present results demonstrate in brief, therefore, that plasma accumulation of the purine catabolic intermediate hypoxanthine may reflect an impaired fetal physiological state, i.e. imbalances in acid-base status, before the onset of labor in growth-retarded fetuses.
Hypoxanthine is formed and released from many tissues of the body, including the brain, heart, and liver and, in the fetus, by the placenta (20, 21). During hypoxia and acidemia, it is derived largely from degradation of adenosine and inosine monophosphate (22) in many tissues, including the ischemic placenta (23). The finding of local regions of infarction and ischemia in placentas of the present study is consistent with placental release of adenosine and its breakdown products and their subsequent release into the fetal circulation.
It is now well established that growth-retarded fetuses are at increased risk of intrauterine death, fetal distress during labor, and increased perinatal mortality. Because this study was performed before the onset of labor and there was an elapse of 4.8 ± 0.9 weeks between the time of cordocentesis and delivery, we could not establish whether there was a direct relationship between umbilical venous levels and fetal prognosis. However, elevated hypoxanthine levels in the fetoplacental circulation, we speculate, may be causally related to these increased risks because of direct tissue damage from oxygen-derived free radicals produced by the action of xanthine oxidase (24). Clearly these speculations await further experimental investigation.
In summary, the present results are consistent with the hypothesis that the elevation of umbilical venous hypoxanthine concentration is a reflection of an unphysiological fetal state, as indicated by fetoplacental acidosis. Our observations suggest that growth-retarded fetuses are exposed to a potentially dangerous environment in utero. Additional intrapartum complications, such as preeclampsia and intrauterine infection, may lead to an increased risk of perinatal morbidity and mortality.
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Acknowledgments |
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Footnotes |
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Received May 12, 1997.
Revised July 30, 1997.
Accepted August 26, 1997.
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References |
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