Thiele, Ingrit G. I. MD*; Niezen-Koning, Klary E. PhD†; van Gennip, Albert H. PhD‡; Aarnoudse, Jan G. MD, PhD*
There is convincing evidence that the various clinical manifestations of preeclampsia are the result of endothelial dysfunction.1,2 In this respect the possible role of oxidative stress in the pathophysiology of preeclampsia has attracted much attention.3 The presence of elevated lipid hydroperoxides, reduced antioxidants, and dyslipidemia suggests that abnormalities in the fatty acid metabolism exist in preeclampsia.4 Also early supplementation with antioxidants (vitamins C and E) may be effective in decreasing oxidative stress and preventing preeclampsia as shown in a subgroup of women identified by uterine artery Doppler screening.5
The possible role of abnormal lipid metabolism, in particular, abnormal fatty acid oxidation, in the pathophysiology of preeclampsia was recently demonstrated: 79% of the pregnant women who are carrying a fetus with a long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, a defect in the mitochondrial fatty acid oxidation, develop the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, a severe consequence of preeclampsia.6 This is most likely the result of toxic metabolites originating from the long-chain 3-hydroxyacyl-CoA dehydrogenase–deficient placenta entering the maternal circulation. This finding is also of interest because it is generally accepted that the placenta is a key factor in the pathophysiology of preeclampsia and is thought to be the main source of factors that eventually lead to endothelial cell dysfunction in the mother.7,8
Fatty acid oxidation plays a major physiological role during tissue energy depletion because of fasting, febrile illness, or increased muscular activity. Fatty acids are used as a major metabolic fuel by the human placenta.9,10 Fatty acids, as their CoA-ester, are transported into the mitochondrion over the mitochondrial membrane, where oxidation of fatty acids occurs. Carnitine is a critical factor for the transport of long-chain fatty acids into the mitochondrion.11–13 In the mitochondrial matrix, fatty acids of various lengths (short-chain, medium-chain, and long-chain fatty acids) are available as their acyl-CoA- esters, ready to undergo β-oxidation. When acyl-CoA esters accumulate because of either a defect or a diminished activity in fatty acid β-oxidation, they will be converted to their corresponding carnitine ester. This ester can leave the mitochondria and can be detected as acylcarnitine in blood. Total carnitine consists of free carnitine and its esters. In the plasma, most of the esterified carnitine is present as acetylcarnitine. The total number of esters of carnitine in plasma is referred to as acylcarnitine.
Plasma carnitine analysis is an important diagnostic tool for recognizing deficiencies in fatty acid oxidation. It has been shown that plasma carnitine concentrations decrease during normal pregnancy.14–17 The importance of carnitine in fatty acid oxidation during tissue energy depletion, combined with the lower plasma carnitine concentrations during pregnancy, led us to hypothesize that the abnormal fatty acid metabolism in preeclampsia is associated with a decrease in plasma carnitine concentration. The aim of the study was to evaluate the possible role of abnormal fatty acid oxidation in preeclampsia by comparing plasma carnitine levels between preeclamptic and healthy control pregnant women.
MATERIALS AND METHODS
The study was approved by the medical ethics committee of the University Hospital Groningen. Recruitment of patients and healthy control subjects was done at the antenatal ward and the antenatal clinic of the University Hospital Groningen. During a period of 7 months, all women hospitalized for preeclampsia at the antenatal ward of the department were approached for recruitment. The controls were randomly identified during prenatal visits at the low-risk antenatal clinic during the same period when the patients were recruited. Women were included if the following criteria were met: singleton pregnancies and preeclampsia according to the criteria of the International Society for the Study of Hypertension in Pregnancy.
Preeclampsia was defined according to the standards of the International Society for the Study of Hypertension in Pregnancy: a diastolic blood pressure (Korotkov V) of at least 90 mm Hg on 2 or more consecutive occasions, each more than 4 hours apart, and proteinuria 300 mg/24 hours, or at least 2+ on dipstick. Sometimes these features were accompanied by complaints such as generalized edema, headache, visual disturbance, nausea and vomiting, and epigastric pain. Excluded were multiple pregnancies and women with preexistent hypertension, diabetes, renal dysfunction, or immune diseases and women with intrauterine fetal death. Controls were healthy pregnant women without hypertension or proteinuria. Neither patients nor control subjects were in labor when studied.
After giving their informed consent, 33 women with preeclampsia and 30 healthy pregnant control subjects were included. Two control patients developed pregnancy hypertension in the last few weeks of their pregnancy and were therefore excluded; the remaining 28 control subjects had an uneventful pregnancy after the time of blood sampling. Blood was collected by venipuncture and centrifuged, and the plasma was stored at −80°C. The plasma carnitine and acylcarnitines were investigated with electrospray tandem mass spectrometry.18 Briefly, this is a fast, specific, and sensitive technique for analyzing polar, nonvolatile, biological molecules. Electrospray tandem mass spectrometer consists of an ion source, commonly a quadropole mass spectrometer, and a conventional detector. The quadropole mass spectrometer consists of 3 mass filters connected in tandem. The molecules are ionized by electrospray ionization. The ions formed are filtered by the mass spectrometer and passed through the detector. The reproducibility of electrospray tandem mass spectrometry for plasma carnitine assessment was previously tested, and coefficients of variation of 8.6% and 6–15% for free carnitines and the different acylcarnitines, respectively, were found.18 For a more detailed description of plasma acylcarnitine analysis by electrospray tandem mass spectrometry, the reader is referred to the literature.18
All samples were analyzed in the same batch. To enable good analysis of the results, the various fractions of acylcarnitines were put together in 3 different groups: short-, medium-, and long-chain acylcarnitines. The total acylcarnitine concentrations were a sum of the short-, medium-, and long-chain acylcarnitine concentrations. These concentrations plus the free carnitine concentrations revealed the total carnitine concentrations.
From the results of a previous study in our laboratory of healthy adults (Albert H. van Gennip, personal communication, 2002), we calculated that, to detect a 30% difference in acylcarnitine concentrations, at least 25 women with preeclampsia and 25 controls were required (α = .5 and β = .20). Maternal blood samples of the preeclampsia and the control groups were tested for differences with the double-sided t test for unpaired samples. The χ2 test was used to analyze differences in nulliparity and cesarean delivery rate between patients and controls. Correlations with maternal variables were calculated by using the Pearson correlation. Differences, with P < .05, were regarded statistically significant.
Table 1 shows the most relevant clinical data. Women were similar with respect to gestational age at sampling. The percentage of nulliparous women was higher in the preeclampsia group, but the difference was not statistically significant. The lower birth weight, together with the lower gestational age at birth and the high caesarean delivery rate, underline the severity of the disease in the preeclamptic group.
In the preeclampsia group, all the plasma carnitine concentrations were significantly increased, except for the medium-chain acylcarnitines (Table 2). Compared with the control group, values were increased by approximately half.
Total and short-chain plasma acylcarnitine levels were significantly correlated to diastolic blood pressure, with correlation coefficients of 0.38 (P < .01) and 0.36 (.01< P < .02), respectively. No statistical relationship could be demonstrated between carnitine concentrations and the variables proteinuria and systolic blood pressure.
Carnitine plays an indispensable role in fatty acid metabolism.12,13 Carnitine is essential for the transport of fatty acids over the inner and outer mitochondrial membranes into the mitochondrion, where β-oxidation takes place. Besides, carnitine has a role in removing excess toxic metabolites, eg, acyl groups, which are excreted as acylcarnitines by the kidneys. Carnitine homeostasis depends on dietary intake, synthesis in the liver and kidneys, and tubular reabsorption.12,13
Studies in normal pregnancies have shown a decrease in plasma-free carnitine and total carnitine levels, the decrease being more pronounced during the first half of pregnancy.14–16 At delivery, maternal plasma carnitine levels are approximately half of those seen in nonpregnant women.17 In the present study, we also found a trend of decreasing plasma carnitine levels toward term in the preeclampsia group as well as in the control group. However, in our study the decrease was not statistically significant. The decrease in plasma carnitine concentrations during normal pregnancy is not fully understood. The decrease cannot solely be explained by the physiological hemodilution of pregnancy.15 It has been suggested that transplacental transfer to supply the fetus might be partly responsible for the decrease in maternal plasma carnitines.17 Carnitine is actively transported across the placenta by an organic cation/carnitine transporter (OCTN2) that is highly expressed in the placenta.19 Also, an increased need to remove excess acylgroups in pregnancy has been mentioned as an explanation for the lower plasma carnitines in pregnancy.17
In the present study, we found that, in the preeclampsia group, plasma carnitine concentrations were increased by approximately 50% compared with the control group. This was true for the free carnitines as well as for the acylcarnitines, and thus for the total carnitines. Therefore, we had to reject our hypothesis that was based on the presumption that the available carnitine for fatty acid oxidation was reduced as a possible explanation for the disturbed fatty acid oxidation in preeclampsia. We found 1 study from 1981, in which increased levels of total plasma carnitines were found in 8 pregnant women with “essential hypertension.”14 Also of interest is the finding that elevated levels of acetylcarnitine were found in amniotic fluid of pregnancies complicated by “toxemia of pregnancy,” as defined by proteinuria, edema, and hypertension.20
There are several possible explanations for the increased plasma carnitine concentrations in preeclampsia. Because preeclampsia is often accompanied by a diminished circulating plasma volume, hemoconcentration occurs. However, the degree of hemoconcentration that can be found in preeclampsia cannot fully explain the 50% increase in plasma carnitine concentration in the present study.
Recent studies have shown the activity and expression of various enzymes involved in fatty acid oxidation in the human placenta.9,10 These findings indicate that fatty acids undergo extensive mitochondrial β-oxidation in the placenta and are used as a significant metabolic fuel and energy source for the placenta.10 In the case of disturbed fatty acid oxidation in the placenta, accumulation of abnormal metabolites, eg, acylcarnitines, occurs. This has been demonstrated in a number of inheritable fatty acid oxidation enzyme disorders including long-chain 3-hydroxyacyl-CoA dehydrogenase, where the affected fetus may cause significant morbidity in the mother, which is at least partly ascribed to harmful metabolites originating from the long-chain 3-hydroxyacyl-CoA dehydrogenase–deficient, fetal-derived placenta.10 These abnormal metabolic precursors, including acylcarnitines, are likely transferred to the maternal circulation and cause tissue damage leading to preeclampsia and liver damage,10 a central feature of the HELLP syndrome. The accumulation of toxic metabolites might also induce increased lipid peroxidation and a decrease in antioxidants in the placenta.9,21 Free radicals will flow into the maternal circulation, causing tissue damage and leading to preeclampsia.9
Abnormal placentation, eg, absent or restricted physiological dilatation of the spiral arteries, is a key factor in the pathophysiology of early-onset preeclampsia. As a consequence, uteroplacental blood flow cannot meet the increasing demand of the growing fetus, and placental ischemia occurs.22 This obviously has an impact on lipid metabolism of the placenta, as shown by studies from decidua basalis tissue and in vitro studies in which increased production of lipid hydroxyperoxides was found.23,24 As a possible explanation for our finding of increased plasma acylcarnitines we suggest that, in the abnormal placenta of preeclampsia, fatty acid oxidation is also disturbed. On the analogy of the long-chain 3-hydroxyacyl-CoA dehydrogenase–deficient placenta, the toxic metabolites, including acylcarnitines and lipid hydroperoxides, enter the maternal circulation affecting endothelial vascular and other tissues.25 These toxic metabolites could also be transferred to the fetal circulation, and increased levels of total carnitine and long-chain acylcarnitines have been found immediately after birth in the cord blood of neonates with severe perinatal asphyxia.26
Reduced placental transfer of nutrients to the fetus, resulting in a low birth weight, was present in a number of the subjects in the preeclamptic study group, and this might also affect the transfer of free carnitines, thus explaining the increase in maternal free plasma carnitines in preeclampsia.
In view of the reported association between fetal fatty acid oxidation enzyme defects and maternal liver disease in pregnancy,6,11,25 we were particularly interested in the maternal plasma carnitine concentrations in this group. However, the small number of women with a HELLP syndrome (n = 6) in the preeclampsia group so far does not enable us to make any conclusion on this issue.
Could a disturbed renal function explain the increased plasma carnitines in preeclampsia? Abnormal renal function in preeclampsia is characterized by a reduction in renal blood flow and glomerular filtration rate, whereas glomerular permeability increases and tubular reabsorption is impaired.27 Although, in our preeclampsia group, no severe renal abnormal functioning, eg, oliguria or anuria, was found, we cannot exclude the possibility that a reduced renal clearance of carnitines and acylcarnitines occurred.
The percentage of nulliparous women was higher in the control group than in the preeclamptic group. To exclude a possible effect of parity on plasma carnitine levels, we examined the effect of parity in the control group. Because no significant differences in plasma carnitine levels were found in this group, the effect of parity is unlikely to explain our findings in the preeclampsia group. Finally, one can speculate that the oxidative stress or dyslipidemia of preeclampsia could stimulate the genes involved in the carnitine biosynthesis.
Although the mechanisms causing endothelial dysfunction have not been fully clarified, there is growing evidence that oxidative stress could be an important factor.1,2 Oxidative stress is known to occur in abnormal lipid metabolism. In preeclampsia, increased lipid peroxidation products from the placenta, as well as dyslipidemia, have been demonstrated to be associated with markers of oxidative stress. Thus, abnormal lipid metabolism could be a key factor in the mechanism leading to endothelial dysfunction in preeclampsia. Carnitine and its acylester play an essential role in lipid transport and in metabolic processes, including fatty acid oxidation.
Our findings of the considerable increased plasma carnitine concentrations, together with the known accumulation of lipids, support the hypothesis of abnormal fatty acid metabolism in the pathophysiology of preeclampsia. The possible contribution of disturbed fatty acid oxidation in the placenta needs further investigation.
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