- Email: [email protected]
Studies on asphyxia: Lipids in the alveoli of rats in hypoxic state
215
Forensic Science International, 42 (19891215-220 Elsevier Scientific Publishers Ireland Ltd.
STUDIES HYPOXIC
ON ASPHYXIA: STATE
HIROKI TAKAHASHP, NORIKO TABATAb
LIPIDS
IN THE ALVEOLI
OF RATS
IN
YOSHIO KUROKI’, MASAHIKO MORITAb and
“Dept. of Biochemistry (Section 1) and bDept. of Legal Medicine, Sapporo Medical College, Niahi 17 Minami 1. Chuo-ku, Sapporo 060 lJapad (Received September 21st, 1988) (Revision received November 8th, 19881 (Accepted November 14th. 19881
Summary Biochemical characterization in alveolar lavage fluids of rats which had inhaled 5% oxygen,
carbon dioxide and carbon monoxide was studied in comparison with control rats. The protein content, consisting mainly of serum albumin, markedly increased in the hypoxic states. The phospholipid content also increased one and a half to two times as much as in the control rats. The phospholipids which increased in the alveoli were mainly pulmonary surfactant phospholipids, i.e. phosphatidylcholine and phosphatidylglycerol. The phospholipid profiles did not appear to be affected by the leakage of plasma lipids. These findings indicate that pulmonary surfactant phospholipids accumulate in the alveoli of rats in the hypoxic states examined here. Key wordu: Asphyxia; Pulmonary surfactant; Hypoxia; Lipids; Alveolar lavage
Introduction
In published reports on the morphological changes in rat lungs in the hypoxic state Morita and Tabata [1,2] pointed out that many lattice-formed structures and/or tubular myelin and the membraneous structures appeared in a large amount of the homogenous substance in the alveolar space of the 5O/6Oz-group rats, with no such substance in the 10% O,-group. Similar results were found in other experiments [3,4] in which such gases as carbon monoxide, carbon dioxide and methane were used. The appearance of both the lattice-formed structures and/or tubular myelin, and the debris-like fine structures which appeared as a liquid-like homogeneous substance under light microscopy were the same as those in the previous experiments [1,2]. As mentioned in the previous reports, based on the morphological findings, it was speculated that the debris-like fine structures are likely to have a close relation to the lattice-formed structures and/or tubular myelin. Many reports have been published on the chemical analysis of the alveolar 03794738/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
216
lavage in humans [4,5] as well as animals age in these reports imply a “pulmonary on the alveolar surface detectable by the This paper reports the results of the rats in hypoxic states.
[6-81. The components of the lavsurfactant” existing physiologically analysis of alveolar lavage. analysis of the alveolar lavage of
Materials and Methods Animal
Wistar albino male rats weighing m200-300 g were used for the control group, 5% oxygen group, and the carbon monoxide and dioxide groups in this experiment. Gas concentration
Gas concentrations for the respiration of rats were 5O/b oxygen and 95% nitrogen for the 5%~group and ordinary air for the control group and the same concentrations as stated in the previous reports [2,3] for the carbon monoxide and dioxide groups. Gases for all groups except the control group were prepared by using a fluorometer and were drawn into an observation box into which the rats and the gas concentrations were checked by gas were placed, chromatography as reported in the previous paper [1,2]. Lavage of lung Washing of the alveolar
wall. The whole lung with trachea was excised immediately after the death of rats, and after weighing, washing of the alveolar wall was performed as follows. A polyethylene tube, Hibiki, size 6, connected with a 5-ml syringe was inserted into the trachea and fixed firmly by ligation. Physiological saline was gently poured into the lung through a polyethylene tube and then the fluid was sucked out from the lung as much as possible. Washing was repeated five times with fresh saline solution each time so as to obtain approximately 25 ml lavage. Washing of the lung. The lungs of control rats were also washed after death by Nembutal overdose injection into the abdomen. All lavages were refrigerated at - 80 OC- - 84OC.
Biochemical analysis Protein analysis. Protein
content in the lavage was determined by the method of Lowry et al. [9]. The proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis according to the method of Laemmli [lo]. The protein bands were stained with Coomassie Brilliant Blue. Lipid analysis. Lipids in the lavage were extracted by the method of Bligh and Dyer [ll]. Individual phospholipids were analyzed by two-dimensional
217
thin-layer chromatography as described by Poorthuis et al. [12] using boric acid impregnated silica gel G plates. Lipid phosphorus was determined by the method of Bartlett [13]. Results and Discussion The content of protein in the alveolar spaces of rats inhaled 5% O,, carbon dioxide and carbon monoxide increased markedly compared to that of control rats, i.e. the amounts of protein in these hypoxic groups were six to seven times that of the control group (Table 1). As shown in Fig. 1, the main protein component which increased in these hypoxic states was serum albumin. These findings indicate that plasma albumin leakages into alveolar spaces occur in those hypoxic states induced by 5% 0,, carbon dioxide and carbon monoxide. The phospholipid content also increased in the alveolar spaces of rats in hypoxic states. The increase in the phospholipid content was one and half to two times as much as in the control group (Table 1). The phospholipid composition in the lavage of rats in the hypoxic states were similar to that of the control rats (Table 2). The main phospholipids in the alveolar spaces were phosphatidylcholine (approx. 75 -790/b) and phosphatidylglycerol (approx. 9 - 11’S), which are well known to be characteristic phospholipids of pulmonary surfactant. The content of individual phospholipids in the alveolar spaces were found to have increased significantly in the hypoxic states examined (Table 3). The phospholipid profiles in the hypoxic states did not appear to be affected by the leakage of plasma lipids, which contain much sphingomyelin and lysophosphatidylcholine. These results, therefore, indicate that pulmonary surfactant phospholipids increase in the alveolar spaces of rats in the hypoxic states. From the biochemical viewpoint, the results in the present study strongly support the morphological evidence previously reported by us [l-3] which showed the accumulation of pulmonary surfactant in the hypoxic states.
TABLE
1
CONTENT
OF PROTEIN
Values are mean f
AND PHOSPHOLIPID
C0?&01 In = 41 Protein (mglg wet tissue) Phospholipid (mglg wet tissue)
IN RAT ALVEOLAR
LAVAGE
S.D. (n). ?? P < 0.01.
2.17 f
0.16
0.96 + 0.16
5 % Oxygen ln = 71
Carbon dioxide In = 51
Carbon monoxide In = 41
11.2
11.4
11.2
-c 3.0*
1.62 -c 4.0*
r 2.8*
1.87 2 0.08*
2 1.4*
1.54 + 0.06*
Fig. 1. SDS-polyacrylamide gel electrophoresis of proteins in alveolar lavage fluids of rats. Each 40 pg of proteins was applied on the gels. Protein bands were stained by Coomassie Brilliant Blue. Lane 1, marker proteins; lane 2, crude pulmonary surfactant fraction isolated from alveolar lavage fluids of untreated rats. 36K and 34K proteins are pulmonary surfactant-associated proteins; lanes 3 and 4, untreated; lanes 5 and 6, treated by carbon dioxide; lanes 7 and 8, treated by carbon monoxide; lanes 9 and 10, treated by 5% oxygen. The arrow represents albumin band.
TABLE 2 PHOSPHOLIPID
COMPOSITION OF THE RAT ALVEOLAR
WASH (mol %l
Values are mean f S.D. (nl -: not detected.
Phosphatidylcholine ethanolamine glycerol inositol serine > Sphingomyelin Lysophosphatidylcholine Lyso-bis-phosphatidic acid
Control In = 41
5% Oxygen fn = 7/
Carbon
Carbon
dioxide In = 51
monoxide ln = 41
78.2 f 5.2 2.9 + 1.1 10.9 + 2.2
79.6 + 4.2 3.9 f 1.4 9.3 f 0.8
78.8 + 5.2 3.9 f 1.2 9.1 * 1.1
75.2 f 3.9 4.4 f 1.2 8.5 f 1.2
1.1 f 0.6
2.4 f 1.5
3.5 -c 2.7
5.4 f 1.7
3.5 f 1.0 1.9 2 0.8 -
3.0 f 0.7 2.8 2 2.0 -
3.0 f 0.8 _
3.0 2 0.8 2.2 2 1.3
219
. TABLE 3 CONTENT OF INDIVIDUAL
PHOSPHOLIPIDS
IF “T
ALVEOLAR
LAVAGE (rmol)
Values are mean * S.D. (n). -: not detected. In = -4)
5% Oxygen h&n=?).
Carbon diode In = 51
Carbon monoxide In = 4)
Phosphatidylcholine ethanolamine glycerol inositol serine 1
0.75 f 0.05 0.03 f 0.01 0.11 f 0.01
1.29 + 0.07 0.06 + 0.02 0.15 f 0.01
1.47 f 0.10 0.07 & 0.02 0.17 f 0.02
1.15 r 0.06 0.07 f 0.02 0.13 f 0.02
0.01
0.04 * 0.02
0.05 + 0.02
0.08 f 0.03
Sphingomyelin Lysophosphatidylcholine
0.03 f 0.01 0.02 f 0.01
0.05 f 0.01 0.05 + 0.03
0.06 f 0.02 -
0.04 f 0.01 -
Co7&ol
Lyso-biiphosphatidic
acid
-
-
-
0.03 2 0.02
Acknowledgements The authors are indebted to the instruction and encouragement given by Professor T. Akino, Chief of the Dept. of Biochemistry, Section 1. They also wish to express their thanks to Miss Atsuyo Maya for the preparation of materials and in the analytical work. This work was supported in part by the Grant-in-Aid (61570294) for Scientific Research from the Ministry of Education, Science and Culture of Japan. Referencea M. Morita, N. Tabata and A. Maya, Studies on asphyxia: on the changes of alveolar wall of rate in hypoxic state. Forensic Sci Ink, 27 (1985) 81-92. M. Morita and N. Tabata, Studies on asphyxia: on the changes of alveolar wall of rats in hypoxic state. II. The hypoxic state produced by carbon dioxide and methane. Forensic Sci Znt., 39 (1988) 267 - 262. M. Morita and N. Tabata, Studies on asphyxia: on the changes of alveolar wall of rats in hypoxic state. III. The hypoxic state by carbon monoxide gas. Unpublished. J. F&mire&, B. Schwartz, A&. Dowell and SD. Lee, Biochemical composition of human pulmonary washing. Arch. Intern Med, 127 (1971) 395-400. T. Onodera, M. Nakamura, W. Sate and T. Akino, Biochemical characterization of pulmonary washings of patients with alveolar proteinosis, interstitial pneumonitis and alveolar cell carcinoma. Tohoku J. Exp. Med, 139 (1983) 246-263. N. Toshima and T. Akino, Alveolar and tissue phospholipids of rat lung. Tohoku J. Exp. Med, 108 (1972) 253- 263. T. Fujiwara, F.H. Adams, S. Sipos and A. El-Salawy, “Alveolar” and whole lung phospholipids of the developing fetal lamb lung. Am. J. Physiol. 215 (1964) 375-35”. T.E. Morgan, T.N. Finley and H. Failkow, Comparison of the composition and surface activity of “alveolar” and whole lung lipids in dog. Biochim. Biopftys. Acta, 106 (1965) 430- 413. D.H. Lowry, N.J. Bosenbrough, A.L. Farr and R.T. Randall, Protein measurements with the folin phenol reagent. J. BioL, Chem.. 193 (1951) 265 - 275.
220
10
U.K. Laemmli.
Cleavage
of structural
proteins
during
the head of bacteriophage
T4.
Nature, 227 (1970) 680 - 685.
11
E.G. Bligh and W.J. Dyer, A rapid method of total lipid extraction and purification. Can J. 37 (1959) 911-917. B.J.H.M. Poorthuis, P.J. Yazaki and K.Y. Hostetler, An improved two dimensional thinlayer chromatography system for the separation of phosphatidylglycerol and its derivatives. J. Lipid Res., 17 (1976) 433-437. W.C. Bartlett, Phosphorus assay in column chromatography. J. Biol Chem., 234 (1959) 466-
Biochem. Physiol,
12
13
468.
Forensic Science International, 42 (19891215-220 Elsevier Scientific Publishers Ireland Ltd.
STUDIES HYPOXIC
ON ASPHYXIA: STATE
HIROKI TAKAHASHP, NORIKO TABATAb
LIPIDS
IN THE ALVEOLI
OF RATS
IN
YOSHIO KUROKI’, MASAHIKO MORITAb and
“Dept. of Biochemistry (Section 1) and bDept. of Legal Medicine, Sapporo Medical College, Niahi 17 Minami 1. Chuo-ku, Sapporo 060 lJapad (Received September 21st, 1988) (Revision received November 8th, 19881 (Accepted November 14th. 19881
Summary Biochemical characterization in alveolar lavage fluids of rats which had inhaled 5% oxygen,
carbon dioxide and carbon monoxide was studied in comparison with control rats. The protein content, consisting mainly of serum albumin, markedly increased in the hypoxic states. The phospholipid content also increased one and a half to two times as much as in the control rats. The phospholipids which increased in the alveoli were mainly pulmonary surfactant phospholipids, i.e. phosphatidylcholine and phosphatidylglycerol. The phospholipid profiles did not appear to be affected by the leakage of plasma lipids. These findings indicate that pulmonary surfactant phospholipids accumulate in the alveoli of rats in the hypoxic states examined here. Key wordu: Asphyxia; Pulmonary surfactant; Hypoxia; Lipids; Alveolar lavage
Introduction
In published reports on the morphological changes in rat lungs in the hypoxic state Morita and Tabata [1,2] pointed out that many lattice-formed structures and/or tubular myelin and the membraneous structures appeared in a large amount of the homogenous substance in the alveolar space of the 5O/6Oz-group rats, with no such substance in the 10% O,-group. Similar results were found in other experiments [3,4] in which such gases as carbon monoxide, carbon dioxide and methane were used. The appearance of both the lattice-formed structures and/or tubular myelin, and the debris-like fine structures which appeared as a liquid-like homogeneous substance under light microscopy were the same as those in the previous experiments [1,2]. As mentioned in the previous reports, based on the morphological findings, it was speculated that the debris-like fine structures are likely to have a close relation to the lattice-formed structures and/or tubular myelin. Many reports have been published on the chemical analysis of the alveolar 03794738/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
216
lavage in humans [4,5] as well as animals age in these reports imply a “pulmonary on the alveolar surface detectable by the This paper reports the results of the rats in hypoxic states.
[6-81. The components of the lavsurfactant” existing physiologically analysis of alveolar lavage. analysis of the alveolar lavage of
Materials and Methods Animal
Wistar albino male rats weighing m200-300 g were used for the control group, 5% oxygen group, and the carbon monoxide and dioxide groups in this experiment. Gas concentration
Gas concentrations for the respiration of rats were 5O/b oxygen and 95% nitrogen for the 5%~group and ordinary air for the control group and the same concentrations as stated in the previous reports [2,3] for the carbon monoxide and dioxide groups. Gases for all groups except the control group were prepared by using a fluorometer and were drawn into an observation box into which the rats and the gas concentrations were checked by gas were placed, chromatography as reported in the previous paper [1,2]. Lavage of lung Washing of the alveolar
wall. The whole lung with trachea was excised immediately after the death of rats, and after weighing, washing of the alveolar wall was performed as follows. A polyethylene tube, Hibiki, size 6, connected with a 5-ml syringe was inserted into the trachea and fixed firmly by ligation. Physiological saline was gently poured into the lung through a polyethylene tube and then the fluid was sucked out from the lung as much as possible. Washing was repeated five times with fresh saline solution each time so as to obtain approximately 25 ml lavage. Washing of the lung. The lungs of control rats were also washed after death by Nembutal overdose injection into the abdomen. All lavages were refrigerated at - 80 OC- - 84OC.
Biochemical analysis Protein analysis. Protein
content in the lavage was determined by the method of Lowry et al. [9]. The proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis according to the method of Laemmli [lo]. The protein bands were stained with Coomassie Brilliant Blue. Lipid analysis. Lipids in the lavage were extracted by the method of Bligh and Dyer [ll]. Individual phospholipids were analyzed by two-dimensional
217
thin-layer chromatography as described by Poorthuis et al. [12] using boric acid impregnated silica gel G plates. Lipid phosphorus was determined by the method of Bartlett [13]. Results and Discussion The content of protein in the alveolar spaces of rats inhaled 5% O,, carbon dioxide and carbon monoxide increased markedly compared to that of control rats, i.e. the amounts of protein in these hypoxic groups were six to seven times that of the control group (Table 1). As shown in Fig. 1, the main protein component which increased in these hypoxic states was serum albumin. These findings indicate that plasma albumin leakages into alveolar spaces occur in those hypoxic states induced by 5% 0,, carbon dioxide and carbon monoxide. The phospholipid content also increased in the alveolar spaces of rats in hypoxic states. The increase in the phospholipid content was one and half to two times as much as in the control group (Table 1). The phospholipid composition in the lavage of rats in the hypoxic states were similar to that of the control rats (Table 2). The main phospholipids in the alveolar spaces were phosphatidylcholine (approx. 75 -790/b) and phosphatidylglycerol (approx. 9 - 11’S), which are well known to be characteristic phospholipids of pulmonary surfactant. The content of individual phospholipids in the alveolar spaces were found to have increased significantly in the hypoxic states examined (Table 3). The phospholipid profiles in the hypoxic states did not appear to be affected by the leakage of plasma lipids, which contain much sphingomyelin and lysophosphatidylcholine. These results, therefore, indicate that pulmonary surfactant phospholipids increase in the alveolar spaces of rats in the hypoxic states. From the biochemical viewpoint, the results in the present study strongly support the morphological evidence previously reported by us [l-3] which showed the accumulation of pulmonary surfactant in the hypoxic states.
TABLE
1
CONTENT
OF PROTEIN
Values are mean f
AND PHOSPHOLIPID
C0?&01 In = 41 Protein (mglg wet tissue) Phospholipid (mglg wet tissue)
IN RAT ALVEOLAR
LAVAGE
S.D. (n). ?? P < 0.01.
2.17 f
0.16
0.96 + 0.16
5 % Oxygen ln = 71
Carbon dioxide In = 51
Carbon monoxide In = 41
11.2
11.4
11.2
-c 3.0*
1.62 -c 4.0*
r 2.8*
1.87 2 0.08*
2 1.4*
1.54 + 0.06*
Fig. 1. SDS-polyacrylamide gel electrophoresis of proteins in alveolar lavage fluids of rats. Each 40 pg of proteins was applied on the gels. Protein bands were stained by Coomassie Brilliant Blue. Lane 1, marker proteins; lane 2, crude pulmonary surfactant fraction isolated from alveolar lavage fluids of untreated rats. 36K and 34K proteins are pulmonary surfactant-associated proteins; lanes 3 and 4, untreated; lanes 5 and 6, treated by carbon dioxide; lanes 7 and 8, treated by carbon monoxide; lanes 9 and 10, treated by 5% oxygen. The arrow represents albumin band.
TABLE 2 PHOSPHOLIPID
COMPOSITION OF THE RAT ALVEOLAR
WASH (mol %l
Values are mean f S.D. (nl -: not detected.
Phosphatidylcholine ethanolamine glycerol inositol serine > Sphingomyelin Lysophosphatidylcholine Lyso-bis-phosphatidic acid
Control In = 41
5% Oxygen fn = 7/
Carbon
Carbon
dioxide In = 51
monoxide ln = 41
78.2 f 5.2 2.9 + 1.1 10.9 + 2.2
79.6 + 4.2 3.9 f 1.4 9.3 f 0.8
78.8 + 5.2 3.9 f 1.2 9.1 * 1.1
75.2 f 3.9 4.4 f 1.2 8.5 f 1.2
1.1 f 0.6
2.4 f 1.5
3.5 -c 2.7
5.4 f 1.7
3.5 f 1.0 1.9 2 0.8 -
3.0 f 0.7 2.8 2 2.0 -
3.0 f 0.8 _
3.0 2 0.8 2.2 2 1.3
219
. TABLE 3 CONTENT OF INDIVIDUAL
PHOSPHOLIPIDS
IF “T
ALVEOLAR
LAVAGE (rmol)
Values are mean * S.D. (n). -: not detected. In = -4)
5% Oxygen h&n=?).
Carbon diode In = 51
Carbon monoxide In = 4)
Phosphatidylcholine ethanolamine glycerol inositol serine 1
0.75 f 0.05 0.03 f 0.01 0.11 f 0.01
1.29 + 0.07 0.06 + 0.02 0.15 f 0.01
1.47 f 0.10 0.07 & 0.02 0.17 f 0.02
1.15 r 0.06 0.07 f 0.02 0.13 f 0.02
0.01
0.04 * 0.02
0.05 + 0.02
0.08 f 0.03
Sphingomyelin Lysophosphatidylcholine
0.03 f 0.01 0.02 f 0.01
0.05 f 0.01 0.05 + 0.03
0.06 f 0.02 -
0.04 f 0.01 -
Co7&ol
Lyso-biiphosphatidic
acid
-
-
-
0.03 2 0.02
Acknowledgements The authors are indebted to the instruction and encouragement given by Professor T. Akino, Chief of the Dept. of Biochemistry, Section 1. They also wish to express their thanks to Miss Atsuyo Maya for the preparation of materials and in the analytical work. This work was supported in part by the Grant-in-Aid (61570294) for Scientific Research from the Ministry of Education, Science and Culture of Japan. Referencea M. Morita, N. Tabata and A. Maya, Studies on asphyxia: on the changes of alveolar wall of rate in hypoxic state. Forensic Sci Ink, 27 (1985) 81-92. M. Morita and N. Tabata, Studies on asphyxia: on the changes of alveolar wall of rats in hypoxic state. II. The hypoxic state produced by carbon dioxide and methane. Forensic Sci Znt., 39 (1988) 267 - 262. M. Morita and N. Tabata, Studies on asphyxia: on the changes of alveolar wall of rats in hypoxic state. III. The hypoxic state by carbon monoxide gas. Unpublished. J. F&mire&, B. Schwartz, A&. Dowell and SD. Lee, Biochemical composition of human pulmonary washing. Arch. Intern Med, 127 (1971) 395-400. T. Onodera, M. Nakamura, W. Sate and T. Akino, Biochemical characterization of pulmonary washings of patients with alveolar proteinosis, interstitial pneumonitis and alveolar cell carcinoma. Tohoku J. Exp. Med, 139 (1983) 246-263. N. Toshima and T. Akino, Alveolar and tissue phospholipids of rat lung. Tohoku J. Exp. Med, 108 (1972) 253- 263. T. Fujiwara, F.H. Adams, S. Sipos and A. El-Salawy, “Alveolar” and whole lung phospholipids of the developing fetal lamb lung. Am. J. Physiol. 215 (1964) 375-35”. T.E. Morgan, T.N. Finley and H. Failkow, Comparison of the composition and surface activity of “alveolar” and whole lung lipids in dog. Biochim. Biopftys. Acta, 106 (1965) 430- 413. D.H. Lowry, N.J. Bosenbrough, A.L. Farr and R.T. Randall, Protein measurements with the folin phenol reagent. J. BioL, Chem.. 193 (1951) 265 - 275.
220
10
U.K. Laemmli.
Cleavage
of structural
proteins
during
the head of bacteriophage
T4.
Nature, 227 (1970) 680 - 685.
11
E.G. Bligh and W.J. Dyer, A rapid method of total lipid extraction and purification. Can J. 37 (1959) 911-917. B.J.H.M. Poorthuis, P.J. Yazaki and K.Y. Hostetler, An improved two dimensional thinlayer chromatography system for the separation of phosphatidylglycerol and its derivatives. J. Lipid Res., 17 (1976) 433-437. W.C. Bartlett, Phosphorus assay in column chromatography. J. Biol Chem., 234 (1959) 466-
Biochem. Physiol,
12
13
468.