- Email: [email protected]
Blood isoprene concentrations in humans and in some animal species
BIOCHEMKAL
MEDICINE
AND
METABOLIC
BIOLOGY
47, 157-160 (1992)
Blood lsoprene Concentrations in Humans and in Some Animal Species ANNIE
CAILLEUX,*
MARC COGNY~ AND PIERRE ALLAIN*
*Laboratoire de Pharmcologie, Centre Hospitalier Universitaire, Rue Larrey, 49033 Angers cedex 01, France; and tEcole Nationale V&&inaire, Service de Physiologic, CP 3013, 44021 Nantes ceder 03, France
Received December 10, 1991 The concentrations of isoprene, the main hydrocarbon of human breath, were measured in the blood of humans and of different animal species (rat, rabbit, dog, ewe, cow). In human blood, the concentrations of isoprene were between 15 and 70 nmol/liter (mean value of 37 -t 25 (SD) nmol/liter). In the blood of the different animal species tested, traces of isoprene were unambiguously detected by mass spectrometry, but the levels were always lower than 1 nmol/liter o 1992 Academic press. h.
Isoprene (2-methyl-1,3-butadiene) is the main hydrocarbon of human breath (l-4). A circadian variation with a maximum concentration during the night was described by DeMaster and Nagasawa (5). In a previous study (6), we showed that isoprene concentration depended on states of sleep and wakefulness rather than on a circadian rhythm. The origin of isoprene is not clearly known, but it is probably produced from mevalonic acid (MVA), a precursor of cholesterol (7,8). The concentration of isoprene in blood has not yet been determined. Our purpose in this study was to measure blood isoprene concentration in humans and in different animal species, with the eventual aim of identifying the animal organs producing isoprene . MATERIAL
AND METHODS
Human. Ten milliliters of blood were collected in the morning from 10 volunteers, 6 women and 4 men, ages 30 to 50 years. Subjects were not fasting and were awake. Animals. Blood samples were collected from 2 rats, 2 rabbits, 4 equidae (ponies), 5 dogs, 5 bovidae, and 7 sheep. Animals were awake and were not anesthetized. Blood was taken from the jugular veins of all animal species except the rat, where blood was sampled from the heart. Metho& Isoprene was quantified using methods previously described (9). Ten milliliters of blood was collected directly into a 25-ml heparinized glass vial with 157 0885-4505192 $3.00 Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
158
CAILLEUX,
COGNY,
AND ALLAIN
a cap sealed with a Teflon-face silicone-rubber septum. Blood was allowed to equilibrate with the gas phase at 45°C during 10 min before analysis. Then isoprene was extracted from the gas phase of the vial by passing a stream of helium above the sample and concentrated into a tenax trap refrigerated to -50°C by liquid nitrogen. After a purge time of 10 min, the trap was rapidly heated to 280°C and isoprene was flushed into two capillary columns (RSL 160 Alltech) of a gas chromatograph (Varian Model 6000). One of the capillary columns was connected to a flame ionization detector (FID) and the other to a mass spectrometer ion trap detector (ITD 800 Finnigan). The column temperature, after an initial step at 40°C for 5 min, was increased to 200°C at a rate of 10”C/min. The injector and detector were maintained at 250°C. Helium was used as carrier gas at a flow rate of 1 ml/min. The quantification limits using the FID detector and the mass spectrometer at the fragment M = 67 were 5 and 1 nmol/liter, respectively. RESULTS In humans isoprene was detectable with the flame ionization detector and quantification was done using this detector. The concentrations of isoprene in human blood were between 15 and 70 nmol/liter with a mean value of 37 ? 25 (SD) nmol/liter. In the blood of the different species of animals studied (rats, rabbits, ponies, dogs, cows, and ewes) no isoprene peak was detected with the flame ionization detector, but using the ion trap detector, very low levels of isoprene were unambiguously detected (Fig. 1). The sensitivity and specificity of the mass spectrometer are sufficient to confirm that traces of isoprene were present in the blood of the tested animals but not sufficient for precise quantification of the levels, which were lower than 1 nmol/liter. Thus, the concentration of isoprene is more than 30 times higher in human blood than in the blood of the animal species studied. DISCUSSION We did not find in the literature any data on the concentration of isoprene in the blood of humans or animals. The higher concentration of isoprene in human compared to animal blood is well correlated with the results obtained in breath. Several authors (l-5) described isoprene in human breath but not in animal breath. According to Deneris et al. (7) isoprene was only found in breath of suckling rats and its production stopped after weaning. All our experiments were done on adult animals and we do not know if isoprene is present at higher levels in young animals. According to data in the literature (7,8) isoprene synthesis seems linked to the synthesis of mevalonic acid (MVA). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG CoA red) catalyzes the conversion of HMG CoA to MVA which in vitro in a rat liver cytosolic fraction produces isoprene via isopentenyl and dimethylallypyrophosphate formation. MVA is the common precursor of cholesterol, ubiquinone, sterols, and dolichols, and important differences in the levels of these latter products could be observed between animal and human species.
BLOOD
ISOPRENE
loo
IN HUMANS
AND ANIMALS
159
* (b) 53
50
FIG. 1. Mass spectra of isoprene authentic compound (a), isoprene in human blood (b), and isoprene in ewe blood (c). The molecular weight of isoprene is 68. The 53 and 67 fragments correspond to the loss of CH3 and H, respectively. The 56, 61, and 62 fragments in ewe blood arise from mass spectrometer background linked to a very high amplification.
Cholesterol concentrations in blood are lower in animal species than in man. For example, in rats and rabbits, widely used for lipid studies (lo), cholesterol levels in blood are about four times lower than those in human blood. Dolichol also shows differences according to species. In comparison with corresponding organs of the rat, chicken, and pig, human tissues contain higher levels of dolichol and dolichol phosphate and the range of homologues can be different (11). In the rat, dolichol levels increase after birth for some days and then decrease (12,13). This could be an explanation for the presence of higher levels of isoprene in breath of preweaning rats. It is also known that HMG CoA reductase, the
160
CAILLEUX,
COGNY,
AND ALLAIN
main enzyme in mevalonic acid synthesis, undergoes marked changes during preand postdevelopment (14,15), but the mechanisms controlling these changes of activities are not known, Therefore, differences in the concentrations of isoprene and of polyisoprenoid compounds between animals and humans could be related to differences in HMG CoA reductase activities. We did not find in the literature comparative data on the activity of HMG CoA reductase in animal and human tissues, but Devery et al. (10) showed important differences between animal species: in the rat HMG CoA reductase was approximately 1Cfold higher in liver relative to intestine, whereas in the rabbit comparable levels of the enzyme were observed in both tissues. Our objective in beginning this investigation was to find an animal model to progress in the study of isoprene metabolism and to find the main organs responsible for isoprene production. However, our results show that it is difficult to investigate isoprene production in animals because its concentration is so low. REFERENCES 1. Conkle JP, Camp BJ, Welch BE. Trace composition of human respiratory gas. Arch Environ Health 38~290-295, 1975. 2. Jansson BO, Larsson BT. Analysis of organic compounds in human breath by gas chromatographymass spectrometry. J Lab Clin Med 74~961-966, 1969. 3. Gelmont D, Stein RA, Mead JF. Isoprene, the main hydrocarbon in human breath. Biochem Biophys Res Commun 99:1456-1460, 1981. 4. Jones AW. Excretion of low-molecular weight volatile substances in human breath: Focus on ethanol. J Anal Toxic01 9~246-250, 1985. 5. DeMaster EG, Nagasawa HT. Isoprene, an endogenous constituent of human alveolar air with a diurnal pattern of excretion. Life Sci 22~91-97, 1978. 6. Cailleux A, Allain P. Isoprene and sleep. Life Sci 441:877-1880, 1989. 7. Deneris ES, Stein RA, Mead JF. In vitro biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction. Biochem Biophys Res Commun l23:691-696, 1984. 8. Deneris ES, Stein RA, Mead JF. Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction. J Biol Chem 260:1382-1385, 1985. 9. Cailleux A, Turcant A, Allain P, Toussaint D, Gaste J, Roux A. Gas chromatographic analysis of volatile compounds in water and biological samples with an automatic injector. J Chromatogr 391:280-289, 1987. 10. Devery RAM, O’Meara N, Collins PB, Johnson AH, Scott L, Tomkin GH. A comparative study of the rate limiting enzymes of cholesterol synthesis, esterification and catabolism in the rat and rabbit. Comp Biochem Physiol B 87~697-702, 1987. 11. Rip JW, Rupar A, Ravi K, Carroll KK. Distribution, metabolism and function of dolichol and polyprenols. Prog Lipid Res 24:269-309, 1985. 12. James MJ, Kandutsch AA. Evidence for independent regulation of dolichol and cholesterol synthesis in developing mouse brain. Biochim Biophys Acta 619:432-435, 1980. 13. Rust RS, Sakakihara Y, Volpe JJ. Dolichyl phosphate: Rapid increase and predominant form of brain dolichol compounds during early brain development. Dev Neurosci 10~25-33, 1988. 14. Carlson SE, Mitchell AD, Goldfarb S. Sex-related differences in diurnal activities and development of hepatic microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cholesterol 7a-hydroxylase. Biochim Biophys Acta 531:115-124, 1978. 15. Innis SM. Effect of diet during pregnancy and lactation on the activity of HMG CoA reductase in the developing rat. J Nub 118:1177-1183, 1988.
MEDICINE
AND
METABOLIC
BIOLOGY
47, 157-160 (1992)
Blood lsoprene Concentrations in Humans and in Some Animal Species ANNIE
CAILLEUX,*
MARC COGNY~ AND PIERRE ALLAIN*
*Laboratoire de Pharmcologie, Centre Hospitalier Universitaire, Rue Larrey, 49033 Angers cedex 01, France; and tEcole Nationale V&&inaire, Service de Physiologic, CP 3013, 44021 Nantes ceder 03, France
Received December 10, 1991 The concentrations of isoprene, the main hydrocarbon of human breath, were measured in the blood of humans and of different animal species (rat, rabbit, dog, ewe, cow). In human blood, the concentrations of isoprene were between 15 and 70 nmol/liter (mean value of 37 -t 25 (SD) nmol/liter). In the blood of the different animal species tested, traces of isoprene were unambiguously detected by mass spectrometry, but the levels were always lower than 1 nmol/liter o 1992 Academic press. h.
Isoprene (2-methyl-1,3-butadiene) is the main hydrocarbon of human breath (l-4). A circadian variation with a maximum concentration during the night was described by DeMaster and Nagasawa (5). In a previous study (6), we showed that isoprene concentration depended on states of sleep and wakefulness rather than on a circadian rhythm. The origin of isoprene is not clearly known, but it is probably produced from mevalonic acid (MVA), a precursor of cholesterol (7,8). The concentration of isoprene in blood has not yet been determined. Our purpose in this study was to measure blood isoprene concentration in humans and in different animal species, with the eventual aim of identifying the animal organs producing isoprene . MATERIAL
AND METHODS
Human. Ten milliliters of blood were collected in the morning from 10 volunteers, 6 women and 4 men, ages 30 to 50 years. Subjects were not fasting and were awake. Animals. Blood samples were collected from 2 rats, 2 rabbits, 4 equidae (ponies), 5 dogs, 5 bovidae, and 7 sheep. Animals were awake and were not anesthetized. Blood was taken from the jugular veins of all animal species except the rat, where blood was sampled from the heart. Metho& Isoprene was quantified using methods previously described (9). Ten milliliters of blood was collected directly into a 25-ml heparinized glass vial with 157 0885-4505192 $3.00 Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
158
CAILLEUX,
COGNY,
AND ALLAIN
a cap sealed with a Teflon-face silicone-rubber septum. Blood was allowed to equilibrate with the gas phase at 45°C during 10 min before analysis. Then isoprene was extracted from the gas phase of the vial by passing a stream of helium above the sample and concentrated into a tenax trap refrigerated to -50°C by liquid nitrogen. After a purge time of 10 min, the trap was rapidly heated to 280°C and isoprene was flushed into two capillary columns (RSL 160 Alltech) of a gas chromatograph (Varian Model 6000). One of the capillary columns was connected to a flame ionization detector (FID) and the other to a mass spectrometer ion trap detector (ITD 800 Finnigan). The column temperature, after an initial step at 40°C for 5 min, was increased to 200°C at a rate of 10”C/min. The injector and detector were maintained at 250°C. Helium was used as carrier gas at a flow rate of 1 ml/min. The quantification limits using the FID detector and the mass spectrometer at the fragment M = 67 were 5 and 1 nmol/liter, respectively. RESULTS In humans isoprene was detectable with the flame ionization detector and quantification was done using this detector. The concentrations of isoprene in human blood were between 15 and 70 nmol/liter with a mean value of 37 ? 25 (SD) nmol/liter. In the blood of the different species of animals studied (rats, rabbits, ponies, dogs, cows, and ewes) no isoprene peak was detected with the flame ionization detector, but using the ion trap detector, very low levels of isoprene were unambiguously detected (Fig. 1). The sensitivity and specificity of the mass spectrometer are sufficient to confirm that traces of isoprene were present in the blood of the tested animals but not sufficient for precise quantification of the levels, which were lower than 1 nmol/liter. Thus, the concentration of isoprene is more than 30 times higher in human blood than in the blood of the animal species studied. DISCUSSION We did not find in the literature any data on the concentration of isoprene in the blood of humans or animals. The higher concentration of isoprene in human compared to animal blood is well correlated with the results obtained in breath. Several authors (l-5) described isoprene in human breath but not in animal breath. According to Deneris et al. (7) isoprene was only found in breath of suckling rats and its production stopped after weaning. All our experiments were done on adult animals and we do not know if isoprene is present at higher levels in young animals. According to data in the literature (7,8) isoprene synthesis seems linked to the synthesis of mevalonic acid (MVA). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG CoA red) catalyzes the conversion of HMG CoA to MVA which in vitro in a rat liver cytosolic fraction produces isoprene via isopentenyl and dimethylallypyrophosphate formation. MVA is the common precursor of cholesterol, ubiquinone, sterols, and dolichols, and important differences in the levels of these latter products could be observed between animal and human species.
BLOOD
ISOPRENE
loo
IN HUMANS
AND ANIMALS
159
* (b) 53
50
FIG. 1. Mass spectra of isoprene authentic compound (a), isoprene in human blood (b), and isoprene in ewe blood (c). The molecular weight of isoprene is 68. The 53 and 67 fragments correspond to the loss of CH3 and H, respectively. The 56, 61, and 62 fragments in ewe blood arise from mass spectrometer background linked to a very high amplification.
Cholesterol concentrations in blood are lower in animal species than in man. For example, in rats and rabbits, widely used for lipid studies (lo), cholesterol levels in blood are about four times lower than those in human blood. Dolichol also shows differences according to species. In comparison with corresponding organs of the rat, chicken, and pig, human tissues contain higher levels of dolichol and dolichol phosphate and the range of homologues can be different (11). In the rat, dolichol levels increase after birth for some days and then decrease (12,13). This could be an explanation for the presence of higher levels of isoprene in breath of preweaning rats. It is also known that HMG CoA reductase, the
160
CAILLEUX,
COGNY,
AND ALLAIN
main enzyme in mevalonic acid synthesis, undergoes marked changes during preand postdevelopment (14,15), but the mechanisms controlling these changes of activities are not known, Therefore, differences in the concentrations of isoprene and of polyisoprenoid compounds between animals and humans could be related to differences in HMG CoA reductase activities. We did not find in the literature comparative data on the activity of HMG CoA reductase in animal and human tissues, but Devery et al. (10) showed important differences between animal species: in the rat HMG CoA reductase was approximately 1Cfold higher in liver relative to intestine, whereas in the rabbit comparable levels of the enzyme were observed in both tissues. Our objective in beginning this investigation was to find an animal model to progress in the study of isoprene metabolism and to find the main organs responsible for isoprene production. However, our results show that it is difficult to investigate isoprene production in animals because its concentration is so low. REFERENCES 1. Conkle JP, Camp BJ, Welch BE. Trace composition of human respiratory gas. Arch Environ Health 38~290-295, 1975. 2. Jansson BO, Larsson BT. Analysis of organic compounds in human breath by gas chromatographymass spectrometry. J Lab Clin Med 74~961-966, 1969. 3. Gelmont D, Stein RA, Mead JF. Isoprene, the main hydrocarbon in human breath. Biochem Biophys Res Commun 99:1456-1460, 1981. 4. Jones AW. Excretion of low-molecular weight volatile substances in human breath: Focus on ethanol. J Anal Toxic01 9~246-250, 1985. 5. DeMaster EG, Nagasawa HT. Isoprene, an endogenous constituent of human alveolar air with a diurnal pattern of excretion. Life Sci 22~91-97, 1978. 6. Cailleux A, Allain P. Isoprene and sleep. Life Sci 441:877-1880, 1989. 7. Deneris ES, Stein RA, Mead JF. In vitro biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction. Biochem Biophys Res Commun l23:691-696, 1984. 8. Deneris ES, Stein RA, Mead JF. Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction. J Biol Chem 260:1382-1385, 1985. 9. Cailleux A, Turcant A, Allain P, Toussaint D, Gaste J, Roux A. Gas chromatographic analysis of volatile compounds in water and biological samples with an automatic injector. J Chromatogr 391:280-289, 1987. 10. Devery RAM, O’Meara N, Collins PB, Johnson AH, Scott L, Tomkin GH. A comparative study of the rate limiting enzymes of cholesterol synthesis, esterification and catabolism in the rat and rabbit. Comp Biochem Physiol B 87~697-702, 1987. 11. Rip JW, Rupar A, Ravi K, Carroll KK. Distribution, metabolism and function of dolichol and polyprenols. Prog Lipid Res 24:269-309, 1985. 12. James MJ, Kandutsch AA. Evidence for independent regulation of dolichol and cholesterol synthesis in developing mouse brain. Biochim Biophys Acta 619:432-435, 1980. 13. Rust RS, Sakakihara Y, Volpe JJ. Dolichyl phosphate: Rapid increase and predominant form of brain dolichol compounds during early brain development. Dev Neurosci 10~25-33, 1988. 14. Carlson SE, Mitchell AD, Goldfarb S. Sex-related differences in diurnal activities and development of hepatic microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cholesterol 7a-hydroxylase. Biochim Biophys Acta 531:115-124, 1978. 15. Innis SM. Effect of diet during pregnancy and lactation on the activity of HMG CoA reductase in the developing rat. J Nub 118:1177-1183, 1988.