- Email: [email protected]
Conversion of H3-phytol to phytanic acid and its incorporation into plasma lipid fractions in heredopathia atactica polyneuritiformis
Conversion of H3-Phytol to Phytanic Acid and Its Incorporation into Plasma Lipid Fractions in Heredopathia Atactica Polyneuritiformis By W. KAIILKEAND H. WAGENER The incorporation of radioactivity from orally administered tritium-labeled phytol into phytanic acid of plasma triglycerides, phospholipids, and cholesterol esters was studied in a patient with heredopathia atactica polyneuritlformis (Refsum's disease). Radioactivity was very rapidly incorporated into triglycerlde fatty acids. There was a much slower uptake of the label into phospholipid fatty acids. Despite their low content of phytanlc acid, radioactivity was as rapidly incorporated into cholesterol
ester fatty acids as into triglyceride fatty acids. Specific radioactivltles of phytanlc acid showed a similar time course. From this incorporation pattern it is concluded that plasma cholesterol esters and triglycerides act as the main vehicles for phytanlc acid newly synthesized from exogenous phytol. Reduction of chlorophyll intake might be tried for lowering the phytanic acid content in the plasma of patients with heredopathia atactica polyneuritiformis. (Metabolism 15: No. 8, August, 687-693, 1966)
TER T H E DISCOVERY OF considerable amounts of phytanie (3, 7, 11, 15-tetramethylhexadeeanoie) acid in the tissue lipids 1 of a patient suffering from heredopathia atactiea polyneuritiformis (HAP), Refsum's disease, 2 this branched fatty acid was also isolated from serum. 3,4 In the meantime, phytanic acid was found in a total of 12 patients. 5,6 Plasma total fatty acids in these patients contained 3 to 23 per cent of this unusual fatty acid. Phytanic acid was preferentially located in plasma triglyeerides (TG). Phospholipids (PL) contained smaller amounts. Phytanie acid was undeteetable in plasma cholesterol esters (CE) of most patients, in some eases only this lipid fraction showed between 0.5 and 1.5 per cent phytanic acid2 ,7 Total fatty acids of plasma fi-lipoproteins contained twice as much phytanic acid as a-lipoproteins, s The mechanism of phytanic acid accmnulation in HAP is still unknown. Recently it was found that exogenous phytol is transformed to phytanic acid. s,9 In patients with HAP the catabolism of this diterpenoic acid seems to proceed slower than in normals for Steinberg et al. 1~ observed a delay in C~402 production after administration of U-C14-phytol. Eldjarn TM found a reduction of sebacic acid excretion after feeding tricaprin to HAP patients suggesting a defect in co-oxidation. In this communication we report on the incorporation of radioactivity from tritium-labeled phytot into plasma TG, PL and CE h'actions in a patient with HAP. Specific activities from the various plasma lipid fractions were also examined.
A
Received for publication Apr. 22, 1966. W. KA/JLKF, M.D.: University of Heidelberg, Department of Medicine, Ludolf KrehlClinic, Heidelberg, Germany. H. WACEZ~ER,M.D.: University of Heidelberg, Department of Medicine, Ludolf KrehI-Clinic, Heidelberg, Germany. 687
688
KAHLKE AND WAGENER
Fig. 1 . - - P r e p a r a t i v e thin-layer c h r o m a t o g r a p h y of p l a s m a n e u t r a l lipids. (Plates: 200 • 200 mm., silica gel G of 0.6 mm. thickness. C H = cholesterol, T G --. triglyeerides, C E (front) = cholesterol esters.) METHODS Seventy-two mg. of 6N,10,11,14,15-He'-phytol (for synthesis and purification refer to Stoffel and Kahlke 9) corresponding to 1.2 inc. were administered orally to a patient with Refsum's syndrome after an overnight fast. The clinical data of this patient were already described by Harders and Dieckmann. 13 Venous blood samples were obtained at various intervals after ingestion of the radioactive phytol. Citrated plasnra of these samples was dried by lyophilization and plasma total lipids were extracted with boiling chloroformmethanol (2:1, v:v), purified by washing with 0.04 per cent calcium chloride solution, evaporated i n vaeuo, dried, and Weighed. Total lipids were separated into phospholipids (PL) and neutral lipids on a silieie acid-eelite (2:1, w:w) column by elution with chloroform and methanol-chloroform (95:5, v:v), respectively. The neutral lipid fraction was further separated into cholesterol esters (CE), triglycerides (TG) and cholesterol by thin-layer chromatography (Fig. 1). For this purpose 20-25 rag. of the various neutral lipid fractions were applied as a streak on thin-layer plates (200 • 200 mm.) covered with a silica gel G layer of 0.6 mm. thickness. After developing with n-hexane-acetie acid ethyl ester (4:1, v:v) individual lipid fractions were localized under ultraviolet-light. 14 The lipid containing areas were scraped off carefully to avoid contamination. The silica gel,G fractions were collected into small chromatographic cohmms and eluted with chloroform. Each fraction was evaporated, weighed, and its purity checked by thin-layer chromatography. Aliquots of these lipid fractions were transmethylated with 5 per cent methanolie HC1 and then saponified with 0.5 N methanolic NaOH. After careful extraction of unsaponifiable material fatty acids were liberated by acidification. Fatty acid methyl esters were prepared by refluxing with 5 per cent methanolie HC1 containing a small amount of methyl acetate. 15 Fatty acid methyl esters were dissolved in exactly 10 ml. chloroform and aliquots transferred to counting vessels. After evaporation of the solvent the residues were dissolved in counting solution (4 Gin. PPO and 0.3 Gin. dimethyl-POPOP in 1000 ml. toluene) and
PLASMA
LIPID
FRACTIONS
IN
689
HAP
m
m
o
o
o
C16 m
3,7,11,1 5 -.I E TRM, IETHYL - . HEXADECANOIC ACID ~C16 b )
o
m
C18:2 m
m m
..... : ff • . . . . . . . . . . . . . . . . . . . . . . . . . ~ .... ~ ...... Fig. "2.--Plasma fatty acid composition in a patient with heredopathia ataetiea polyneuritiformis (llefsum's disease). Model 872, Packard Instruments, DEGScolumn, 165 C., 65 ml. argon/minute, 500 volts, sensitivitv 1 • l0 8 amperes, 5 minutes/inch. counted in a liquied scintillation spectrometer (Tricarb Model 4000, Packard Instruments). 200-300 ~g. aliquots of the various fatty acid methyl ester fractions were analysed by gasliquid chromatography (Model 872, Packard Instruments, 12 per cent IBP-coluum, 180 X 6 nlm., 150 C., 130 ml. argon/minute, 500 volts, sensitivity 3 • 10 -7 amp., 5 min./ineh). The argon gas leaving the column was divided with a stream splitter in such a way that only 1/7 of the argon gas reached the ionization detector, whereas 6/7 were introduced into counting vials filled with scintillator solution. In this way portions of the effluent gas stream were collected for 1 minute each. Calculation of fatty acid methyl ester composition was done by triangulation. B.ESULTS
Figure 2 shows the separation of total plasma fatty acids by means of gasliquid chromatography. The phytanic acid (C~6b) peak is located between palmitoleie (C 1 6 : 1 ) and stearic acid ( C 1 8 ) . Specific radioaetivities of fatty acid methyl esters originating from TG, PL and CE fractions of plasma samples obtained at various intervals after phytol administration showed characteristic differences (Table 1). Radioactivity was very rapidly incorporated into T G fatty acids reaching a maximum about 4 hours after phytol ingestion. There was a much slower uptake of the label into PL fatty acids with maximum incorporation 30 hours after phytol administration. Despite their low content of phytanie acid (2 per cent) radioactivity was as rapidly incorporated into CE fatty acids as into T G fatty acids. As shown in Figure 3 radioactivity is contained in the phytanie acid fraction and in another fraction which emerges from the column in front of the myristic acid peak. A similar peak is obtained when phytol is transmethylated with 5 per cent methanolie HC1 and subjected to gas-liquid chromatography. In contrast, untreated phytol has a retention time similar to that of methyl linolenate and a radioactivity peak in this area was never found in our studies.
690
KAHLKE AND WAGENER
Table 1.--Phytanic Acid Content, Radioactivity
of Fatty Acid Methyl Ester Fractions and of Phytanic Acid in Plasma Triglycerides, Cholesterol Esters, and Phospholipids after Ingestion of H3-phytol Radioactivity in Counts/ mg./min.
Plasma Lipid Fraction
Phytanic Acid Content in Time A f t e r H3-Phytol % of Total Application in Hours F a t t y Acids
Phytanic Acid Radioactivity in % of Total Radioactivity
F a t t y Acid Methyl Ester Fraction
Phytanic Acid
TG
1 2 3 4 5 8 12 24 30 36 48
27.5 16.0 25.5 14.0 8.0 36.5 19.5 28.5 30.5 46.5 45.0
33 43 40 37 57 74 14 32 21 9 33
862 10972 35324 62559 35266 62911 28545 32555 38959 37780 37290
CE
1 2 3 4 5 8 12
1.46 2.0 1.77 1.09 (1.0) (1.50)
58 58 80 90
214 5036 7067 11772 10415
24
0.91
13
30 36 48
2.12
7
13154 7972 5882
39521
1 2 3 4 5 8 12 24 30 36 48
12.43 8.4 7.62 8.24 8.0 10.95 9.98 10.24 15.65 10.50 10.18
9 21 35 31 39 57 74 77 90 91 74
5800 4199 4143 9039 7897 8889 7042 13336 33511 22721 23698
3991 10663 18717 33484 38878 46079 51975 100000 192709 197490 173399
PL
1100 29500 54300 165350 251250 127550 20500 36550 26800 7300 27350
4761
199340 205891 541220 852162 (231600) 190300
8439
125090
Radioactivity determinations in fatty acid methyl ester fractions after gas chromatographic separation permitted calculation of radioactivity values of the phytanie acid peaks of the various fatty acid methyl ester mixtures. From these values, total radioactivity of weighed methyl ester fractions, and from the proportions of phytanie acid in the different methyl ester mixtures, speeifie radioactivity values of phytanie acid were obtained. As can be seen from Table 1 and Figure 4, the peak specific activities of total fatty acid methyl esters and phytanie acid methyl ester are reached at similar time intervals after phytol ingestion with the phytanic acid specific activity being considerably higher.
69I
PLASlVfA LIPID FRACTIONS IN t I A P
PhytoniAcid c Acetone
16:0
' ~ ~ J ~ ~ 1 8 : 1 690Ipm
2350Ipm
Fig. 3.--Preparative gas chromatographic separation of plasma triglyceride fatty acid methyl esters, 8 hours after HZ-phytol administration. (Hatch columns indicate radioactivity content of corresponding areas.) DISCUSSION
The considerable increase of specific radioactivity of CE and TG phytanie acid at a time when presumably maximum absorption of orally administered labeled phytol takes place demonstrates that phytanie acid in this fraction is predominantly composed of newly synthesized material. Apparently plasma CE and TG contain the bulk of phytanie acid originating from exogenous phytol. Specific activities of phytanic acid in TG show a similar time course as in CE, but the maximum specific activity is only one-third that of CE phytanic acid specific activity. This is probably due to the much greater amounts of unlabeled phytanie acid present in TG fatty acids. Phytanie aeid from plasma PL reaches its maximum specific activity not until 36 hours after phytol ingestion. At this time CE and TG already show lower specific aetivity values. This could result from transfer of newly synthesized phytanie acid from CE and TG fractions to plasma PL. The latter fraction may then determine the further metabolic pathways of phytanie acid. The origin of the radioactivity appearing in the beginning part of the radiogas chromatogram is not known, injection of solvent only into file column never led to radioactivity values exceeding background values. Possibly this material results from administered phytol which may still be present in plasma on the basis of an impaired transformation to phytanic acid, which in turn may be secondary to diminished oxidation of phytanie acid in HAP. 11 Normally, unaltered phytol is found in the unsaponifiable fraction. Because in this experiment treatment with methanolic HC1 was performed prior to saponification a phytol derivative produced in vitro may be present in the final extracts. On the other hand, it is possible that the unknown fraction represents some material resulting from in vivo transformation of phytanie acid, particularly in view of the fact that the amount of this material increases during the experiment. Biosynthesis of phytanic acid from exogenous phytoI besides, perhaps, other
692
KAHLKE AND WAGENER
Specific activity of Dhy|anic acid (dpm/mg)
x 104 90
9
Phosphol i pids
9
Triglycerides
9
Chotesterol esters
(m) See text
80
70
60
50-
40-
30I
9
20
(,)
.,/\
\
/\
' " - _
/"
f_.
0 ' "; . L & < J
+
r
J
234 S
8
12
24
30
36
48 hr$
Fig. 4.--Time course of specific activity of phytanic acid in plasma triglycerides, cholesterol esters, and phospholipids after Ha-phytol ingestion. (Values of cholesterol
ester phytanic acid specific activity at 8 and 12 hours were calculated without gas chromatographic separation of fat@ acid methyl ester mixtures.) precursors supports the assumption that phytanie acid is a substance also present in minor amounts in normals, 5 who ingest phytol as component of chlorophyll present in food. Recent observations by Klenk and Kremer 16,17 on the occurrence of phytanie acid in plasma from normals support this view. Diminished chlorophyll ingestion is presently being evaluated as a possible means for lowering the phytanic acid content in the plasma of the patients with HAP.
PLASMA LIPID FRACTIONS IN HAP
693
ACKNOWLEDGMENT We acknowledge the technical assistance of Miss I. Erbe, Miss S. Brammer and Mrs. E. Bauer. REFERENCES
1. Klenk, E., and Kahlke, W:: fiber das Vorkominen der 3,7,11,I5-Tetramethylhexadeeans~iure (Phytanstiure) in den Cholesterinestern und anderen Lipoidfraktionen der Organe bei einem Krankheitsfall unbekannter Genese (Verdacht anf Heredopathia ataetica polyneuritiformis, RefsumSyndrom). Hoppe-Seylers Z. Physiol. Chem. 333:133, 1963. 2. Refsum, S.: Heredopathia ataetica polyneuritiformis. A familial syndrome not hitherto described. Acta Psyehiat. et Neurol. Stand. Suppl. 38:303, 1946. 3. Kahlke, W.: Ober das Vorkommen von 3, 7, I I, i5- Tetramethylhexadecans~iure fin Blutsermn bei Refsuin-Syndrom. Klin. Wschr. 41:783, 1963. 4. - - , and Richterieh, R.: Refsum's disease (heredopathia atactica polyneuritiformis): An inborn error of lipid metabolism with storage of 3,7,11,15tetramethylhexadeeanoie acid. II. Isolation and identification of the storage produet. Amer. J. Med. 39:237, 1965. 5 . - - : Refsum-Syndrom. Lipoidchemisehe Untersuehungen bei 9 Fallen. Klin. Wsehr. 42:1011, 1964. 6. - - : Unpublished results, 1965. 7. Try, K,, Stokke, D., and Eldjarn, L.: Two new cases of heredopathia ataetica polyneuritiformis (Refsum's disease) demonstrating phytanie acid accumulation. Seand. J. Clin. Lab. Invest. (Suppl. 86) 17:195, 1965. 8. Kahlke, W., and Wagener, H.: Distribution of 3,7,11,15-tetramethylhexadecanoie acid between plasma lipoproreins in Refsmn's syndrome. In H. Peeters (Ed.): Protides of the Biological Fluids, 1965. Amsterdam, London, New York, Elsevier, 1966, p. 351. 9. Stoflel, W., and Kahlke, W.: The transformation of phytol into 3,7,11,15-
I0.
11 .
12.
13.
14.
15.
16.
17.
tetramethylhexadecanoie ( phytanic ) acid in heredopathia atactica polyneuritiformis (Refsmn's syndrome). Bioehem. Biophys. Res. Comm. 19:33, 1965. Steinberg, D., Avigan, ]., Mize, C., and Baxter j.: Phytanic acid formation and accumulation in phytol-fed rats. Biochem. Biophys. Res. Comm. 19: 412, 1965. . . . Eldjarn, L , Try, K., and Refsum S.: Conversion of U-C TM -phytol to phytanie acid and its oxidation in heredopathia ataetiea polyneuritiformis, Bioehem. Biophys. Res. Comm. 19:783, 1965. Eldjarn, L.: Heredopathia ataetica polyneuritiformis (Refsum's disease). A defect in the omega-oxidation mechanism of fatty acids. Seand. J. Clin. Lab. Invest. 17:178, 1965. Harders, H., and Dieckmann, H.: Heredopathia atactiea polyneuritiformis. Klinik und Diagnostik des I/efsumSyndroms. Dtsch. Med. Wschr. 89: 248, 1964. Wagener, H.: Detection and documentation of lipids after thin-layer chromatography. Nature 205:386, I965. Eberhagen, D.: In N. Z611ner and D. Eberhagen ( Eds. ) : Untersuchung und Bestiminung der Lipoide im Blut. Berlin, Heidelberg, New York, Springer-Verlag, 1965, p. 152. Kremer, G. J.: Ober das Vorkommen der 3,7,11,15-Tetramethylhexadecanshure in den Lipoiden von Normalseren. Klin. Wschr. 43:517, 1965. Klenk, E., and Kremer, G. J.: Untersuchungen zmn Stoffwechsel des Phytols, Dihydrophytols und der Phytans~iure. Hoppe-Seylers Z. Physiol. Chem. 343:39, 1966.
ester fatty acids as into triglyceride fatty acids. Specific radioactivltles of phytanlc acid showed a similar time course. From this incorporation pattern it is concluded that plasma cholesterol esters and triglycerides act as the main vehicles for phytanlc acid newly synthesized from exogenous phytol. Reduction of chlorophyll intake might be tried for lowering the phytanic acid content in the plasma of patients with heredopathia atactica polyneuritiformis. (Metabolism 15: No. 8, August, 687-693, 1966)
TER T H E DISCOVERY OF considerable amounts of phytanie (3, 7, 11, 15-tetramethylhexadeeanoie) acid in the tissue lipids 1 of a patient suffering from heredopathia atactiea polyneuritiformis (HAP), Refsum's disease, 2 this branched fatty acid was also isolated from serum. 3,4 In the meantime, phytanic acid was found in a total of 12 patients. 5,6 Plasma total fatty acids in these patients contained 3 to 23 per cent of this unusual fatty acid. Phytanic acid was preferentially located in plasma triglyeerides (TG). Phospholipids (PL) contained smaller amounts. Phytanie acid was undeteetable in plasma cholesterol esters (CE) of most patients, in some eases only this lipid fraction showed between 0.5 and 1.5 per cent phytanic acid2 ,7 Total fatty acids of plasma fi-lipoproteins contained twice as much phytanic acid as a-lipoproteins, s The mechanism of phytanic acid accmnulation in HAP is still unknown. Recently it was found that exogenous phytol is transformed to phytanic acid. s,9 In patients with HAP the catabolism of this diterpenoic acid seems to proceed slower than in normals for Steinberg et al. 1~ observed a delay in C~402 production after administration of U-C14-phytol. Eldjarn TM found a reduction of sebacic acid excretion after feeding tricaprin to HAP patients suggesting a defect in co-oxidation. In this communication we report on the incorporation of radioactivity from tritium-labeled phytot into plasma TG, PL and CE h'actions in a patient with HAP. Specific activities from the various plasma lipid fractions were also examined.
A
Received for publication Apr. 22, 1966. W. KA/JLKF, M.D.: University of Heidelberg, Department of Medicine, Ludolf KrehlClinic, Heidelberg, Germany. H. WACEZ~ER,M.D.: University of Heidelberg, Department of Medicine, Ludolf KrehI-Clinic, Heidelberg, Germany. 687
688
KAHLKE AND WAGENER
Fig. 1 . - - P r e p a r a t i v e thin-layer c h r o m a t o g r a p h y of p l a s m a n e u t r a l lipids. (Plates: 200 • 200 mm., silica gel G of 0.6 mm. thickness. C H = cholesterol, T G --. triglyeerides, C E (front) = cholesterol esters.) METHODS Seventy-two mg. of 6N,10,11,14,15-He'-phytol (for synthesis and purification refer to Stoffel and Kahlke 9) corresponding to 1.2 inc. were administered orally to a patient with Refsum's syndrome after an overnight fast. The clinical data of this patient were already described by Harders and Dieckmann. 13 Venous blood samples were obtained at various intervals after ingestion of the radioactive phytol. Citrated plasnra of these samples was dried by lyophilization and plasma total lipids were extracted with boiling chloroformmethanol (2:1, v:v), purified by washing with 0.04 per cent calcium chloride solution, evaporated i n vaeuo, dried, and Weighed. Total lipids were separated into phospholipids (PL) and neutral lipids on a silieie acid-eelite (2:1, w:w) column by elution with chloroform and methanol-chloroform (95:5, v:v), respectively. The neutral lipid fraction was further separated into cholesterol esters (CE), triglycerides (TG) and cholesterol by thin-layer chromatography (Fig. 1). For this purpose 20-25 rag. of the various neutral lipid fractions were applied as a streak on thin-layer plates (200 • 200 mm.) covered with a silica gel G layer of 0.6 mm. thickness. After developing with n-hexane-acetie acid ethyl ester (4:1, v:v) individual lipid fractions were localized under ultraviolet-light. 14 The lipid containing areas were scraped off carefully to avoid contamination. The silica gel,G fractions were collected into small chromatographic cohmms and eluted with chloroform. Each fraction was evaporated, weighed, and its purity checked by thin-layer chromatography. Aliquots of these lipid fractions were transmethylated with 5 per cent methanolie HC1 and then saponified with 0.5 N methanolic NaOH. After careful extraction of unsaponifiable material fatty acids were liberated by acidification. Fatty acid methyl esters were prepared by refluxing with 5 per cent methanolie HC1 containing a small amount of methyl acetate. 15 Fatty acid methyl esters were dissolved in exactly 10 ml. chloroform and aliquots transferred to counting vessels. After evaporation of the solvent the residues were dissolved in counting solution (4 Gin. PPO and 0.3 Gin. dimethyl-POPOP in 1000 ml. toluene) and
PLASMA
LIPID
FRACTIONS
IN
689
HAP
m
m
o
o
o
C16 m
3,7,11,1 5 -.I E TRM, IETHYL - . HEXADECANOIC ACID ~C16 b )
o
m
C18:2 m
m m
..... : ff • . . . . . . . . . . . . . . . . . . . . . . . . . ~ .... ~ ...... Fig. "2.--Plasma fatty acid composition in a patient with heredopathia ataetiea polyneuritiformis (llefsum's disease). Model 872, Packard Instruments, DEGScolumn, 165 C., 65 ml. argon/minute, 500 volts, sensitivitv 1 • l0 8 amperes, 5 minutes/inch. counted in a liquied scintillation spectrometer (Tricarb Model 4000, Packard Instruments). 200-300 ~g. aliquots of the various fatty acid methyl ester fractions were analysed by gasliquid chromatography (Model 872, Packard Instruments, 12 per cent IBP-coluum, 180 X 6 nlm., 150 C., 130 ml. argon/minute, 500 volts, sensitivity 3 • 10 -7 amp., 5 min./ineh). The argon gas leaving the column was divided with a stream splitter in such a way that only 1/7 of the argon gas reached the ionization detector, whereas 6/7 were introduced into counting vials filled with scintillator solution. In this way portions of the effluent gas stream were collected for 1 minute each. Calculation of fatty acid methyl ester composition was done by triangulation. B.ESULTS
Figure 2 shows the separation of total plasma fatty acids by means of gasliquid chromatography. The phytanic acid (C~6b) peak is located between palmitoleie (C 1 6 : 1 ) and stearic acid ( C 1 8 ) . Specific radioaetivities of fatty acid methyl esters originating from TG, PL and CE fractions of plasma samples obtained at various intervals after phytol administration showed characteristic differences (Table 1). Radioactivity was very rapidly incorporated into T G fatty acids reaching a maximum about 4 hours after phytol ingestion. There was a much slower uptake of the label into PL fatty acids with maximum incorporation 30 hours after phytol administration. Despite their low content of phytanie acid (2 per cent) radioactivity was as rapidly incorporated into CE fatty acids as into T G fatty acids. As shown in Figure 3 radioactivity is contained in the phytanie acid fraction and in another fraction which emerges from the column in front of the myristic acid peak. A similar peak is obtained when phytol is transmethylated with 5 per cent methanolie HC1 and subjected to gas-liquid chromatography. In contrast, untreated phytol has a retention time similar to that of methyl linolenate and a radioactivity peak in this area was never found in our studies.
690
KAHLKE AND WAGENER
Table 1.--Phytanic Acid Content, Radioactivity
of Fatty Acid Methyl Ester Fractions and of Phytanic Acid in Plasma Triglycerides, Cholesterol Esters, and Phospholipids after Ingestion of H3-phytol Radioactivity in Counts/ mg./min.
Plasma Lipid Fraction
Phytanic Acid Content in Time A f t e r H3-Phytol % of Total Application in Hours F a t t y Acids
Phytanic Acid Radioactivity in % of Total Radioactivity
F a t t y Acid Methyl Ester Fraction
Phytanic Acid
TG
1 2 3 4 5 8 12 24 30 36 48
27.5 16.0 25.5 14.0 8.0 36.5 19.5 28.5 30.5 46.5 45.0
33 43 40 37 57 74 14 32 21 9 33
862 10972 35324 62559 35266 62911 28545 32555 38959 37780 37290
CE
1 2 3 4 5 8 12
1.46 2.0 1.77 1.09 (1.0) (1.50)
58 58 80 90
214 5036 7067 11772 10415
24
0.91
13
30 36 48
2.12
7
13154 7972 5882
39521
1 2 3 4 5 8 12 24 30 36 48
12.43 8.4 7.62 8.24 8.0 10.95 9.98 10.24 15.65 10.50 10.18
9 21 35 31 39 57 74 77 90 91 74
5800 4199 4143 9039 7897 8889 7042 13336 33511 22721 23698
3991 10663 18717 33484 38878 46079 51975 100000 192709 197490 173399
PL
1100 29500 54300 165350 251250 127550 20500 36550 26800 7300 27350
4761
199340 205891 541220 852162 (231600) 190300
8439
125090
Radioactivity determinations in fatty acid methyl ester fractions after gas chromatographic separation permitted calculation of radioactivity values of the phytanie acid peaks of the various fatty acid methyl ester mixtures. From these values, total radioactivity of weighed methyl ester fractions, and from the proportions of phytanie acid in the different methyl ester mixtures, speeifie radioactivity values of phytanie acid were obtained. As can be seen from Table 1 and Figure 4, the peak specific activities of total fatty acid methyl esters and phytanie acid methyl ester are reached at similar time intervals after phytol ingestion with the phytanic acid specific activity being considerably higher.
69I
PLASlVfA LIPID FRACTIONS IN t I A P
PhytoniAcid c Acetone
16:0
' ~ ~ J ~ ~ 1 8 : 1 690Ipm
2350Ipm
Fig. 3.--Preparative gas chromatographic separation of plasma triglyceride fatty acid methyl esters, 8 hours after HZ-phytol administration. (Hatch columns indicate radioactivity content of corresponding areas.) DISCUSSION
The considerable increase of specific radioactivity of CE and TG phytanie acid at a time when presumably maximum absorption of orally administered labeled phytol takes place demonstrates that phytanie acid in this fraction is predominantly composed of newly synthesized material. Apparently plasma CE and TG contain the bulk of phytanie acid originating from exogenous phytol. Specific activities of phytanic acid in TG show a similar time course as in CE, but the maximum specific activity is only one-third that of CE phytanic acid specific activity. This is probably due to the much greater amounts of unlabeled phytanie acid present in TG fatty acids. Phytanie aeid from plasma PL reaches its maximum specific activity not until 36 hours after phytol ingestion. At this time CE and TG already show lower specific aetivity values. This could result from transfer of newly synthesized phytanie acid from CE and TG fractions to plasma PL. The latter fraction may then determine the further metabolic pathways of phytanie acid. The origin of the radioactivity appearing in the beginning part of the radiogas chromatogram is not known, injection of solvent only into file column never led to radioactivity values exceeding background values. Possibly this material results from administered phytol which may still be present in plasma on the basis of an impaired transformation to phytanic acid, which in turn may be secondary to diminished oxidation of phytanie acid in HAP. 11 Normally, unaltered phytol is found in the unsaponifiable fraction. Because in this experiment treatment with methanolic HC1 was performed prior to saponification a phytol derivative produced in vitro may be present in the final extracts. On the other hand, it is possible that the unknown fraction represents some material resulting from in vivo transformation of phytanie acid, particularly in view of the fact that the amount of this material increases during the experiment. Biosynthesis of phytanic acid from exogenous phytoI besides, perhaps, other
692
KAHLKE AND WAGENER
Specific activity of Dhy|anic acid (dpm/mg)
x 104 90
9
Phosphol i pids
9
Triglycerides
9
Chotesterol esters
(m) See text
80
70
60
50-
40-
30I
9
20
(,)
.,/\
\
/\
' " - _
/"
f_.
0 ' "; . L & < J
+
r
J
234 S
8
12
24
30
36
48 hr$
Fig. 4.--Time course of specific activity of phytanic acid in plasma triglycerides, cholesterol esters, and phospholipids after Ha-phytol ingestion. (Values of cholesterol
ester phytanic acid specific activity at 8 and 12 hours were calculated without gas chromatographic separation of fat@ acid methyl ester mixtures.) precursors supports the assumption that phytanie acid is a substance also present in minor amounts in normals, 5 who ingest phytol as component of chlorophyll present in food. Recent observations by Klenk and Kremer 16,17 on the occurrence of phytanie acid in plasma from normals support this view. Diminished chlorophyll ingestion is presently being evaluated as a possible means for lowering the phytanic acid content in the plasma of the patients with HAP.
PLASMA LIPID FRACTIONS IN HAP
693
ACKNOWLEDGMENT We acknowledge the technical assistance of Miss I. Erbe, Miss S. Brammer and Mrs. E. Bauer. REFERENCES
1. Klenk, E., and Kahlke, W:: fiber das Vorkominen der 3,7,11,I5-Tetramethylhexadeeans~iure (Phytanstiure) in den Cholesterinestern und anderen Lipoidfraktionen der Organe bei einem Krankheitsfall unbekannter Genese (Verdacht anf Heredopathia ataetica polyneuritiformis, RefsumSyndrom). Hoppe-Seylers Z. Physiol. Chem. 333:133, 1963. 2. Refsum, S.: Heredopathia ataetica polyneuritiformis. A familial syndrome not hitherto described. Acta Psyehiat. et Neurol. Stand. Suppl. 38:303, 1946. 3. Kahlke, W.: Ober das Vorkommen von 3, 7, I I, i5- Tetramethylhexadecans~iure fin Blutsermn bei Refsuin-Syndrom. Klin. Wschr. 41:783, 1963. 4. - - , and Richterieh, R.: Refsum's disease (heredopathia atactica polyneuritiformis): An inborn error of lipid metabolism with storage of 3,7,11,15tetramethylhexadeeanoie acid. II. Isolation and identification of the storage produet. Amer. J. Med. 39:237, 1965. 5 . - - : Refsum-Syndrom. Lipoidchemisehe Untersuehungen bei 9 Fallen. Klin. Wsehr. 42:1011, 1964. 6. - - : Unpublished results, 1965. 7. Try, K,, Stokke, D., and Eldjarn, L.: Two new cases of heredopathia ataetica polyneuritiformis (Refsum's disease) demonstrating phytanie acid accumulation. Seand. J. Clin. Lab. Invest. (Suppl. 86) 17:195, 1965. 8. Kahlke, W., and Wagener, H.: Distribution of 3,7,11,15-tetramethylhexadecanoie acid between plasma lipoproreins in Refsmn's syndrome. In H. Peeters (Ed.): Protides of the Biological Fluids, 1965. Amsterdam, London, New York, Elsevier, 1966, p. 351. 9. Stoflel, W., and Kahlke, W.: The transformation of phytol into 3,7,11,15-
I0.
11 .
12.
13.
14.
15.
16.
17.
tetramethylhexadecanoie ( phytanic ) acid in heredopathia atactica polyneuritiformis (Refsmn's syndrome). Bioehem. Biophys. Res. Comm. 19:33, 1965. Steinberg, D., Avigan, ]., Mize, C., and Baxter j.: Phytanic acid formation and accumulation in phytol-fed rats. Biochem. Biophys. Res. Comm. 19: 412, 1965. . . . Eldjarn, L , Try, K., and Refsum S.: Conversion of U-C TM -phytol to phytanie acid and its oxidation in heredopathia ataetiea polyneuritiformis, Bioehem. Biophys. Res. Comm. 19:783, 1965. Eldjarn, L.: Heredopathia ataetica polyneuritiformis (Refsum's disease). A defect in the omega-oxidation mechanism of fatty acids. Seand. J. Clin. Lab. Invest. 17:178, 1965. Harders, H., and Dieckmann, H.: Heredopathia atactiea polyneuritiformis. Klinik und Diagnostik des I/efsumSyndroms. Dtsch. Med. Wschr. 89: 248, 1964. Wagener, H.: Detection and documentation of lipids after thin-layer chromatography. Nature 205:386, I965. Eberhagen, D.: In N. Z611ner and D. Eberhagen ( Eds. ) : Untersuchung und Bestiminung der Lipoide im Blut. Berlin, Heidelberg, New York, Springer-Verlag, 1965, p. 152. Kremer, G. J.: Ober das Vorkommen der 3,7,11,15-Tetramethylhexadecanshure in den Lipoiden von Normalseren. Klin. Wschr. 43:517, 1965. Klenk, E., and Kremer, G. J.: Untersuchungen zmn Stoffwechsel des Phytols, Dihydrophytols und der Phytans~iure. Hoppe-Seylers Z. Physiol. Chem. 343:39, 1966.