Carnitine Biosynthesis from y-butyrobetaine and from Exogenous Protein-bound 6-N-Trimethyl-~-lysine by the Perfused Guinea Pig Liver

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc Vol. 259, No. 17, Issue of September 10, pp ,19&1 Printed in U.S.A. Carnitine Biosynthesis from y-butyrobetaine and from Exogenous Protein-bound 6-N-Trimethyl-~-lysine by the Perfused Guinea Pig EFFECT OF ASCORBATE DEFICIENCY ON THE IN SITU ACTIVITY OF 7-BUTYROBETAINE HYDROXYLASE* (Received for publication, February 27, 1984) William A. During, Giuseppe Rettura, Eli Seifter, and Sasha Englardg From the Departments of Biochemistry and Surgery, Albert Einstein College of Medicine, Yeshiua University, Bronx, New York The production of carnitine from peptide-bound 6- contrast, to Neurospora crassa (9, lo), N-methylation of lysine N-trimethyl-L-lysine (Lys(Me3)) or 4-N-trimethyl- occurs as a post-translational event in proteins such as myaminobutyrate (y-butyrobetaine) perfused through iso- ocin, actin, histones, and calmodulin (11). Carnitine is then lated guinea pig livers was investigated. [1MethyL3H] synthesized from peptide-bound 6-N-trimethyl-~-lysine resi- Lys(Me3)-labeled agalacto-orosomucoid (AGOR) and dues after their release by protein degradation (12-14). The asialofetuin were rapidly taken up and degraded by biosynthesis of carnitine from Lys(Me3) occurs as follows: the perfused liver. Most of the free Lys(Me3) derived (a) hydroxylation of Lys(Me3) to 3-hydroxy-6-N-trimethyl-~from Lys(Me&AGOR was released unmodified into the lysine (15-18) (b) aldol cleavage of 3-hydroxy-6-N-trimethylperfusion medium. However, Lys(Me3), arising from L-lysine to glycine and 4-N-trimethylaminobutyraldehyde Lys(Me3)-asialofetuin was converted mostly to y-bu- (15, 19) (c) oxidation of the betaine aldehyde to 4-N-trimetyrobetaine and carnitine. y-butyrobetaine added to the perfusion medium was hybroxylated to carnitine thylaminobutyrate (y-butyrobetaine) (20); and (d) hydroxylby the liver at a rate of 2.3 pmol/h. Guinea pigs main- ation of y-butyrobetaine to L-carnitine (21). To investigate tained on an ascorbate-free diet for days showed various aspects of carnitine biosynthesis in the isolated perlowered ascorbate contents all in tissues measured and, fused rat liver, we have utilized methyl-labeled Lys(Me3)- coincidentally, a sharp reduction in carnitine levels in asialofetuin and Lys(Me,)-AGOR that are endocytosed (i.e. kidney, liver, and cardiac, and skeletal muscle. Car- their uptake is receptor-mediated) by liver parenchymal and nitine production from [1,2,3,4-14C]y-butyrobetaine nonparenchymal cells, respectively, and then degraded within and [rnethyz-3h]lys(me3)-asialofetuin was reduced in the lysosomes of these cells (13, 14). Following almost comperfused livers obtained from ascorbate-deficient plete proteolysis of these proteins, 60-70% of the released guinea pigs. Although hydroxylation of y-butyrobe- Lys(Me,) was converted to y-butyrobetaine, carnitine, and its taine to carnitine was effectively depressed in the per- 0-acetylated derivative, while the residual Lys(Me,) was N- fused isolated livers from ascorbate-deficient animals, acetylated or eliminated unchanged from the cells into the hydroxylation of [rnethyl3h]lys(me3)(derived from circulating perfusate. asialofetuin) to [methyl3h]3-hydroxy-6-n-trimethyl- Functions for vitamins Bs and C in the in vivo synthesis of L-lysine was unaffected. Prior administration of ascor- carnitine have been inferred from the known cofactor requirebate to the medium perfusing the isolated livers caused ments of several enzymatic steps in the biosynthetic pathway, carnitine biosynthesis from all precursors examined to in particular the cleavage of 3-hydroxy-6-N-trimethyl-~-lyreturn to control values. sine and the hydroxylations of Lys(Mea) and y-butyrobetaine, respectively. Indeed we have reported that in the perfused rat liver the presence of 1-amino-D-proline, a vitamin Bg antagonist, depressed the total production of y-butyrobetaine, car- L-Carnitine (L-3-hydroxy-4-N-trimethylaminobutyrate) nitine, and its acetylated derivative from protein-bound has long been recognized for its essential role in the transport Lys(Me,) by as much as 60-80% (14). The further observation of long chain fatty acids across the inner mitochondrial mem- brane as a prelude (1-3). The methyl groups of carnitine are derived from L-methionine (4,5), and the nitrogen atom with the 4-carbon chain as a unit originates from L-lysine (6-8). In the rat and presumably other mammals, in * This work was supported by United States Public Health Service Grants 2R01 AM and 5P01 AG from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Present address, Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 North Wolfe, Baltimore, MD To whom correspondence and reprint requests should be addressed that the decreased synthesis of carnitine was accompanied by an equivalent accumulation of 3-hydroxy-6-N-trimethyl-~lysine provided evidence for a requirement of pyridoxal 5 - phosphate in the cleavage of that intermediate into glycine and 4-N-trimethylaminobutyraldehyde (step b, above). Other investigators have shown that carnitine levels in cardiac and The abbreviations used are: Lys(Mes), 6-N-trimethyl-~-lysine; AGOR, agalacto-orosomucoid; Lys(Me,)-AGOR agalacto-orosomucoid containing peptide-bound 6-N-trimethyl-~-lysine. *This suggestion is supported by recent investigations showing that in rat liver, cytosolic serine transhydroxymethylase accounts for over 90% of the cleavage activity of 3-hydroxy-6-N-trimethyl-tlysine, with some additional minor contribution by the corresponding mitochondrial enzyme (R. Stein and S. Englard, manuscript in preparation).

2 skeletal muscle and liver are significantly depressed in guinea pigs maintained on an ascorbate-deficient diet for 4-5 weeks (22-24). For the rat and guinea pig, hydroxylations of Lys(Me3) in kidney and of both Lys(Me3) and y-butyrobetaine in liver are catalyzed by separate a-ketoglutarate-coupled dioxygenases; these require molecular oxygen, ferrous ions, and ascorbate for their assay in vitro (15-18, 21, 25, 26). In experiments designed to determine the effect of ascorbate deficiency on the in vivo activity of the two hydroxylases in the carnitine biosynthetic pathway, Nelson et al. (23) observed a sharp reduction in the capacity of the kidneys of scorbutic Effect of Ascorbate Deficiency Carnitine on Biosynthesis results in Fig. 2 with those in Table 11). guinea pigs to convert injected [methyl- 4C]6-N-trimethylly- Fig. 2 shows the patterns of radiolabeled metabolites apsine to labeled y-butyrobetaine. An effect on liver Lys(Me3) hydroxylase, however, could not be determined since less than 2% of the administered radioactive Lys(Me3) was absorbed by the liver in the 1-h duration of the experiment (compared to an uptake of approximately 20% for the kidneys). On the other hand, with [methyl-14c]y-butyrobetaine, which is readpearing in the liver and perfusate at various time intervals after addition of [methyl-i4c]ly~(me3)-a~ialofetuin to the perfusion medium. Lys(Mea), presumably released by the lysosomal degradation of Lys(Me3)-asialofetuin, was converted to carnitine and carnitine precurors to the extent of 53% of the peptide-bound Lys(Me3) injected. Radiolabeled carnitine was ily taken up by the liver, no differences were noted between evident at 2 h after addition of the glycoprotein and accucontrol and scorbutic animals in the production of [ C] mulated in the liver for the next 2 h. The only labeled carnitine by the liver. carnitine precursors found in the liver over the course of this Inherent difficulties in such experiments with intact animals, due in large measure to the limited transport of exogenous Lys(Me3) into kidneys and liver (27-29), led us to investigate in greater detail the dependence on ascorbate of experiment were Lys(Me3) and y-butyrobetaine. methyl-14clabeled 4-N-trimethylaminobutyraldehyde and 3-hydroxy-6- N-trimethyl-L-lysine were not detected. Unlike the perfused rat liver (13,14), the isolated guinea pig liver did not acetylate carnitine biosynthesis in the isolated perfused guinea pig liver. Such studies of the overall process of carnitine biosynthesis from administered glycoproteins containing radioactive Lys(Me3) allowed us to discern the differential effect of ascorbate deficiency on the two hydroxylases operating within a single organ on the appropriate precursors generated within that same tissue. The results of those investigations are presented here. EXPERIMENTAL PROCEDURES3 RESULTS Carnitine Biosynthesisfrom y-butyrobetaine by the Isolated Perfused -The conversion of 7-butyrobetaine to carnitine by the perfused guinea pig liver is shown in Table I. [MethyL3H]- or [1,2,3,4-4C]-y-Butyrobetaine administered to the perfusion medium was rapidly hydroxylated to carnitine by the liver at a maximal rate of 0.15 gmol/h/g of tissue. That rate although only approximately 50% of the value reported for isolated rat liver cells (361, far exceeds the estimated rate of carnitine synthesis by the liver calculated from a reported daily turnover rate for carnitine in the rat of gmol per 100 g of body weight (37, 38). At 3 h almost all of the y- butyrobetaine added to the pefusion medium was converted to carnitine and its acetylated derivative. Approximately 60% of the carnitine released from the liver into the circulating perfusate was in the form of acetylcarnitine. lated perfused guinea pig liver, Lys(Me&AGOR appeared to Carnitine Biosynthesis from [methyl- 4C]Lys(Me3)-Asialofetuin and [methyl-3h]lys(me3)-agor by the Isolated Perfused Liuer-[[nethyl- 4C]Lys(Me3)-asialofetuin and [methyl- HILys(Me,)-AGOR were rapidly and quantitatively taken up by the perfused liver with a half-life in the perfusate of 7.5 and 6.6 min, respectively (Fig. 1). Two hours after adminis- The Experimental Procedures are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD Request Document No. 84M-608, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. tration of [methyl-14c]lys(me3)-asialofetuin to the perfused liver, 80% of the added I4C-label was recovered as perchloric acid-soluble radioactivity (Fig. 2). On the other hand, under similar conditions of perfusion only 55% of the Imeth~l-~H] Lys(Me3)-AGOR was hydrolyzed to acid-soluble products in 3 h (Table 11). Furthermore, depending on the labeled glyco- protein added to the perfusion medium, the Lys(Me3) residues released by proteolysis were either extensively converted by the liver to carnitine and its immediate precursor or largely excreted unchanged into the circulating perfusate (compare substantial amounts of Lys(Me3) to 2-N-acetyl-6-N-trimethyl-L-lysine. After 5 h significant amounts of carnitine and acetylcarnitine were detected in the perfusion medium. While no significant accumulation of acetylcarnitine was evident in the liver, this metabolite accounted for 50-70% of the total carnitine released by the liver. Similar results were reported previously for the isolated rat liver perfused with either Lys(Me3)-asialofetuin or Lys(Me3)asialo-orosomucoid (13) and, as shown in the present study, for the guinea pig liver perfused with the immediate precursor of carnitine, y-butyrobetaine (Table I). In contrast to Lys(Me,)-asialofetuin, 80% of the Lys(Me3) released by the degradation of Lys(Me&AGOR was not metabolized further but instead was excreted intact from the liver into the perfusate (Table 11). The remaining 20% of the Lys(Me3) residues were converted to carnitine and carnitine precursors (ie. 3-hydroxy-6-N-trimethyl-~-lysine and y-butyrobetaine). The poor utilization of Lys(Me3)-AGOR for carnitine biosynthesis by the isolated guinea pig liver, presumably due to its relatively slower rate of proteolytic degradation and to the large excretion of the released Lys(Me3) residues unchanged into the perfusion medium, is in contrast to the more efficient conversion of that modified glycoprotein to carnitine and y-butyrobetaine by the isolated perfused rat liver (13,14). Accordingly, in our investigations on the effects of ascorbate deficiency on carnitine biosynthesis by the iso- be of limited use. Effect of Ascorbate Deficiency on Tissue Carnitine Levek- Depletion of tissue ascorbate was significant for guinea pigs maintained on the ascorbate-free diet for only days (Table 111). Except for brain, no further reductions in ascorbate levels were observed in all other organs examined when the animals were continued on this diet for an additional days (or a total of days). In addition to weight loss, the ascorbate-deficient animals showed reduced mobility accompanied by weakness and hemorrhaging in the hind limb pressure points., heart, and skeletal muscle carnitine levels have been reported to be significantly decreased in ascorbate-deficient or severely scorbutic guinea pigs (22-24). As shown in Table

3 10766 Effect of Ascorbate Deficiency on Carnitine Biosynthesis Time min 60 TABLE I Analysis of radioactive metabolites following addition of labeled y-butyrobetaine to isolated perfused guinea pig livers At indicated times after addition of either 5.85 pmol of [methyl-3h]y-butyrobetaine(1.99 x lo6 dpm) or 6.0 pmol of [1,2,3,4-"C]y-butyrobetaine(1.25 x 10' dpm) to the perfusion medium of separate isolated livers obtained from guinea pigs maintained on a regular laboratory diet, extracts of the liver and perfusate were prepared and the radiolabeled metabolites separated and quantified by ion-exchange chromatography as described under "Experimental Procedures." The concentrated extracts contained between 75 and 95% of the total radioactivity initially present in the liver and perfusion medium. Summation of the values for y-butyrobetaine, carnitine, and acetylcarnitine are shown in brackets. Radiolabeled y-butyrobetaine administered 120 [methyl-3h]y-butyrobetaine 180 [methyl3h]y-butyrobetaine a ND. not detected. 5.2 y-butyrobetaine Carnitine Acetylcarnitine % of radioactiue y-butyrobetaine added 40.2 Perfusate Total [73.1] ND" Perfusate Total [79.9] Perfusate Total [80.3] Perfusate Total IO 20 TIME (MINI FIG. 1. Clearance by isolated guinea pig livers of [methyl- 'H]Lys(Me3)-asialofetuin and [rnethyz-sh]lys(me3)-agor present in the perfusing medium, s from normal animals maintained on a regular laboratory diet were excised and perfused by a procedure like that used for rat liver (33) as described under "Experimental Procedures." At various times after addition of ImethyZ-3H]Lys(Me3)-asialofetuin (100 pg, 1.80 X lo6 dpm; A) or [methyl-3h1lys(me3)-agor (85 pg, 7.90 X lo6 dpm; 0) to the perfusion medium, 0.2-ml aliquots of the perfusate were taken and their radioactive contents determined. The clearance curves represent the average of four experiments with asialofetuin and two experiments with AGOR. The inset shows a plot of the log of the perfusate radioactivity versus time; and from the slopes of the straight lines the half-times of clearance for the methylated glycoproteins were calculated. 111, reduction in tissue carnitine content depended on the length of time the animals had been maintained on the ascorbate-free diet. With time, carnitine levels in kidney, liver, heart, and skeletal muscle decreased as much as 20, 53, 45, and 63%, respectively, while those in serum and brain were unchanged. Effect of Ascorbate Deficiency on Carnitine Biosynthesis from [1,2,3,4-14C]y-Butyrobetaine and [methyl-3h]lys(med- l b 4 20 o_ W a l TIME (hr) FIG. 2. Radiolabeled metabolites of Lys(Me3) present in the isolated liver and perfusion medium at various times after addition of [methyz-14c]lys(mes)-asialofetuin. s from normal guinea pigs maintainedon a regular laboratory diet were cyclically perfused with the labeled glycoprotein as described under "Experimental Procedures." At various times after addition of [methyl-"c] Lys(Me,)-asialofetuin (100 pg, 1.75 X lo6 dpm) to the perfusion medium, the radiolabeled components present in extracts of the liver and perfusate were analyzed by ion-exchange chromatography. The results from the liver (a) and the perfusate (b) are expressed as the percentages of peptide-bound [methyl-"c]lys(me3) injected that in turn corresponded to 79.5% of the total radioactivity added. The metabolites include: Lys(Me3) (A), 2-N-acetyl-6-N-trimethyllysine (A), y-butyrobetaine (O), carnitine (O), and acetylcarnitine (0). Asialofetuin by the Isolated Perfused -By utilizing [1,2,3,4-'4C]r-butyrobetaine and [methyl-3h]lys(mes)-asialofetuin we were able simultaneously to investigate the ca-

4 Effect of Ascorbate Deficiency on Carnitine Biosynthesis TABLE I1 Amlysis of radioactive metabolites present in the liver and perfusate at 3 h after addition of [methyl-3hjlys(me3)- AGOR to isolated perfused guinea pig livers Radiolabeled metabolites present in the liver and perfusate were measured at 3 h after addition of 85 pg of [methyl3h]lys(me3)-ag0r(7.9 X 10' dpm) to the perfusion medium of separate isolated livers obtained from animals maintained on a regular laboratory diet. The values obtained are expressed as the percentages of peptidebound [methyl3h]lys(me3) injected (84.3% of the total protein radioactivity). Summation of the values for the five metabolites are shown in brackets. 6-N-Trimethyl- 3-Hydroxy-6-N- y-butyrotrimethyl- L-lysine betaine L-lysine Carnitine % of radioactive peptide-bound Lys(Me3 added Experiment % of [meth~l-~h] Lys(Me3)AGOR Perfusate NDb hydrolyzed" [55.7] Total Experiment % of 0.8 [mth~l-~h] Lys(Me3)-AGOR Perfusate 32.8 hydrolyzed" Total As determined from the radioactivity recovered as acid-soluble counts in the combined liver and perfusateconcentrated extracts. ND. not detected. TABLE I11 Tissue ascorbate and carnitine contents of guinea pigs maintained on an ascorbate-free diet Control animals were fed the ascorbate-free diet but on alternate days received on the average of 1.8 mg/day of ascorbate orally. Values for ascorbate are expressed in terms of micrograms/g wet weight, of tissue or per 100 ml of serum. Carnitine was determined following base hydrolysis of perchloric acid extracts of the various tissues. Concentrations, expressed in terms of nanomoles per g wet weight, of tissue or per ml of serum, therefore represent the summation of tissue-free carnitine and acid-soluble acylcarnitine content. Values for both ascorbate and carnitine are means f S.E. with the number of animals involved in each determination given in Darentheses. ~~ ~ Ascorbate-deficient animals Tissue Control animals days days Ascorbate Serum Brain Heart Skeletal muscle (gastrocnemius) Adrenal glands Spleen Carnitine Serum Brain Kidney Heart Skeletal muscle (gastrocnemius) Significantly lower than control group p < * Significantly lower than control group p < Significantly lower than control group p < f 16.0 (10) 33.8 f 3.0 (9) f 9.5 (10) 23.1 f 2.6 (10) 11.8 f 0.6 (lo) f 91.0 (9) f 13.0 (9) 38.9 f 3.1 (11) f 30.2 (11) f 40.1 (10) f 39.8 (11) f 72.3 (10) f 45.4 (11) f 3.8 (6)" 26.7 f 1.8 (5) 19.7 f 3.9 (5)" 8.3 f 0.4 (5)" 7.0 f 0.7 (5)" 67.4 f 6.9 (5)" 28.4 f 2.5 (5)" 39.7 f 3.6 (7) f 25.2 (9) f 29.0 (9) f 37.7 (9)b f 77.7 (9)' f 66.0 (9)b f 6.4 (8)" 14.8 f 1.7 (8)" 16.0 f 1.9 (8)" 8.8 f 0.6 (8)" 7.7 f 0.9 (8)" 85.2 f 13.4 (8)" 28.4 f 1.9 (8)" 30.1 f 2.9 (10) f 33.4 (IO) f 17.1 (10)' f 24.2 (10)" f 93.6 (10)" f 55.5 (9)" pacity of the isolated perfused liver from control and ascorbate-deficient guinea pigs to hydroxylate added y-butyrobetaine and Lys(Me3) released from Lys(Me&asialofetuin. s from control and ascorbate-deficient guinea pigs were derived from the corresponding groups of animals in Table 111. The radiolabeled metabolites, present in the combined perfusate and liver extracts 3 h after addition of Lys(Me3)- asialofetuin and 2 h after addition of y-butyrobetaine to the perfused liver, were analyzed as described; the results are shown in Tables IV and V. Ascorbate deficiency had no apparent effect on the perfusate clearance of [rneth~l-~h] Lys(Me&asialofetuin (tliz = 5-12 min); and 3 h after addition of the labeled glycoprotein, 80% was recovered as acid-soluble radioactivity. s used as controls were obtained from guinea pigs maintained on the ascorbate-free diet with oral feedings of sodium ascorbate on alternate days (average dose was 1.8 mg/ day) (Table 111). The conversions to carnitine of [1,2,3,4-"C] y-butyrobetaine and [rnethyl-3h]lys(me3)-asialofetuin by these control livers (Tables IV and V) were identical to those observed previously in livers from "normal" untreated guinea pigs maintained on a regular laboratory diet (refer to Table I and Fig. 2). In contrast, carnitine production from [1,2,3,4-14C]y-butyrobetaine and [methyl-3h]lys(me3)-asialofetuin in livers of ascorbate-deficient animals was depressed by 51 and 25%, respectively, and was accompanied by an increased accumulation in the livers of either [1,2,3,4-I4C]- or [methyl- 3H]y-butyrobetaine, but not of [rnethyl-3h]lys(me3). Thus, for the ascorbate-deficient animals as much as 38 and 25% of the radioactivities added as [1,2,3,4-14C]y-butyrobetaine and

5 10768 Effect of Ascorbate Deficiency on Carnitine Biosynthesis TABLE IV been isolated from human, rabbit, and rat livers (40-43) and Effect of ascorbate deficiency on the synthesis of carnitine from recognized in mouse liver (44). To our knowledge, the demy-butyrobetaine by the isolated perfused guinea pig liver onstration in the present study that isolated guinea pig livers Two hours after addition of [1,2,3,4- C]y-butyrobetaine (1.25 X efficiently cleared.-ever 90% of administered methyl-labeled IO6 dpm) to the separate isolated livers already cyclically perfused for 1 h with [methyz-3h]lys(me3)-asialofetuin(table V), [ Cly-buty- Lys(M&)-asialofetuin and Lys(Me,)-AGOR, at rates comparobetaine,carnitine,andacetylcarnitinepresent in the liverand rable to those reported for the rat (13), represents the first perfusion medium were analyzed as described under Experimental evidence that similar recognition sites for receptor-mediated Procedures. The number of livers perfused per experimental group endocytosis occur in guinea pig hepatocellular membranes. is given in parentheses. Values are presented as the means f S.E. of While the rate of hydrolysis of Lys(Me3)-asialofetuin to yield the three metabolites present in the combined liver and perfusate acid-soluble products corresponded closely to that found for extracts. rat liver, degradation of endocytosed Lys(Me3)-AGOR by Radiolabeled Control Scorbutic 'ius guinea pig liver was considerably slower. Because the liver ascorbate metabolites (4) (4) (3) does not take up significant amounts of free Lys(Me3) (27- % of [ Cly-butyrobetaine added 29), in the present investigations we have utilized peptidey-butyrobetaine 13.1 & & 5.9 bound Lys(Me3) which is taken up by the hepatic cells and Carnitine 67.0 f f 6.2 converted to free Lys(Me3); this provided a convenient mech- Acetylcarnitine 2.7 f f 1.7 anism to distinguish the role of hepatocytes and non-paren- Sum 82.8 f f 4.6 chymal cells in carnitine production from Lys(Me3) released a Significantly higher than control group p < intracellularly. Significantly lower than control group p < For ascorbate-deficient or severely scorbutic guinea pigs, significant decreases in liver, heart, and skeletal muscle carnitine contents have been reported (22-24). For the group of peptide-bound [~nethyl-~h]lys(me~), respectively, were recovered in the livers as y-butyrobetaine. Pre-perfusion of livers obtained from ascorbate-deficient guinea pigs with SOdium ascorbate (4 mg/100 ml) for 1 h resulted in a sharp reduction of the hepatic levels of [ 1,2,3,4-14C]y-butyrobetaine (7% of the injected radioactivity) and of [methyl-3h]y-butyrobetaine (4% of the radioactivity added as peptide-bound Lys(Me,)), and increased the production of carnitine from those precursors to values observed with control livers (Tables IV and V). DISCUSSION Receptor-mediated endocytosis has been recognized as the first step in the heterophagy of glycoproteins. Reticuloendothelial cells (Kupffer cells and alveolar macrophages) in the rat and hepatocytes in the chicken possess surface receptors that bind terminal mannose and N-acetylglucosamine residues of glycoproteins (e.g. AGOR) (39). On the other hand, cell surface receptors that specifically recognize and bind galactose-terminating glycoproteins (e.g. asialofetuin) are found only in mammalian liver parenchymal cells and have ascorbate-deficient guinea pigs used in the present investigations, carnitine content of kidney was also significantly decreased but brain and serum contents remained nearly unchanged. A reduction in carnitine contents could decrease the capacity of cells to oxidize fatty acids for provision of energy (1,2). Although the literature is meager in this regard, ascorbate deficiency in guinea pigs is known to produce some effects on lipid metabolism consistent with decreased carnitine synthesis, e.g. diversion of fatty acids from oxidation to excess accumulation and/or formation of triglycerides (22, 45, 46). The activity of proline, 8-oxoglutarate dioxygenase (EC ), the enzyme that catalyzes the hydroxylation of proline residues in procollagen chains, has been shown to be considerably reduced in scorbutic animals, and restored fol- lowing addition of ascorbate (47). In the carnitine biosynthesis pathway, hydroxylation of 6-N-trimethyl-L-lysine and of y-butyrobetaine are catalyzed by separate a-ketoglutaratedependent dioxygenases, but each required addition of ferrous ions and ascorbate to allow expression of enzymatic activity in vitro (15-18, 21, 25, 26). That cofactor requirement sug- TABLE V Effect of ascorbate deficiency on the production of carnitine from [methyl-3h]lysfme~-ain~fetuin by the isolated perfused guinea pig liver Three hours after addition of [methyl-3h]lys(me3)-asialofetuin( pg, X IO6 dpm) to isolated perfused guinea pig livers, the 3H-labeled metabolites present the in liver and perfusion medium were analyzed as described under Experimental Procedures. It is to be noted as indicated in the legend of Table IV, that at 1 h after addition of the labeled glycoprotein, [ C]y-butyrobetaine was added to the perfusion medium and the experiment terminated 2 h later. Values are presented as the means f S.E. of the metabolites present in the combined liver and perfusate extracts and expressed as the percentages of peptide-bound [rnethyl-3h]lys(me3) injected (75.0% of the total protein radioactivity). The number of experimental determinations per group of animals are in parentheses. Summation of the values for the three metabolites that arise following the cleavage of [methyl3h]3-hydroxy-6-n-trimethyllysine are shown in brackets. Radiolabeled Control Scorbutic metabolites (4) (4) Scorbutic plus ascorbate (3) % of peptide-bound [methyl-3hllys(me,j added 2-N-Acetyl-6-N-trimethyllysine 3.8 f k f N-Trimethyllysine 39.7 f f f Hydroxy-6-N-trimethyllysine 1.4 & f ? 0.3 y-butyrobetaine 6.9 f f & 1.2 Carnitine 36.7 f f f 5.0 Acetylcarnitine 1.0 f rt f 0.1 Sum 88.6 f [ f f [ f f [ f 2.11 Significantly higher than control group p < * Significantly lower than control group p < 0.05.

6 ~ ~... Effect of Ascorbate Deficiency gested a possible link between induced ascorbate deficiency in the guinea pig and the observed concomitant pronounced decreases in tissue contents of carnitine. Indeed, Nelson et al. (23) have reported that following injection of [methyl-'4c] Lys(Mes) into the inferior vena cavae of anesthetized guinea pigs, the kidneys of either pair-fed or ad libitum-fed control animals appeared to produce [14C]y-butyrobetaine at rates of 8-10 times greater than those of kidneys of scorbutic animals. The decreased production of y-butyrobetaine from Lys(Me3) suggested that kidney Lys(Me3) hydroxylase activity was sensitive to ascorbate levels. In separate experiments, ascorbate deficiency had no apparent effect on the production of ["C] carnitine from injected [ methyl-'4c]y-butyrobetaine, seeming to indicate that ascorbate had no great significance for the in vivo hydroxylation of y-butyrobetaine by the liver. It should be noted, however, that no decreases in liver carnitine content occurred in either the "pair-fed" or even the scorbutic guinea pigs used in the studies of Nelson et al. (23). That is in contrast to the results of Sandor et al. (24) and those of the present st~dy.~ The contribution and interdependence of individual organs in the overall process of carnitine biosynthesis are difficult to assess in whole animal experiments because of the variable and limited transport into various organs of some carnitine precursors, particularly Lys(Me3) and 3-hydroxy-6-N-trimethyl-L-lysine (23, 27-29), the high rate of flux of carnitine and perhaps also of y-butyrobetaine between liver and blood, and the rapid although variable redistribution of carnitine into different extrahepatic tissues (37, 38, 48). Such considerations, possibly complicated further by unknown effects of ascorbate on those parameters, favored the isolated perfused guinea pig liver as the experimental model to investigate the effects of ascorbate deficiency on carnitine biosynthesis. Furthermore, simultaneous addition of [methyl-3h]lys(me3)-asi- alofetuin and [1,2,3,4-'4C]y-butyrobetaine to the perfusion medium allowed us to discern, for each of the perfused livers, the effects of ascorbate depletion and replenishment on both Lys(Me3) and y-butyrobetaine hydroxylases operating in concert within a single tissue. Of the recovered radioactivity administered as [1,2,3,4-'4C]y-butyrobetaine, 15.8% was present as y-butyrobetaine and 84.2% appeared as carnitine for the control group of animals, while the corresponding values were 51.3 and 48.7% for the ascorbate-deficient animals. Preperfusion of the ascorbate-deficient livers with ascorbate resulted in complete restoration of the capacity to convert y- butyrobetaine to carnitine. For the same livers perfused with [rnethyl-3h]lys(me3)-asialofetuin, ascorbate deficiency did not cause decreased production of metabolites beyond the step of Lys(Me3) hydroxylation, namely, y-butyrobetaine + caritine + acetylcarnitine. [3H]Carnitine, however, represented only 52.7% of the total radioactivity in that pool of metabolites compared to a corresponding value of 82.3% for the controls and 85.4% for the ascorbate-deficient livers preperfused with ascorbate. Thus, for livers of ascorbate-deficient guinea pigs, reduction in carnitine production from [methyl- 'HH]Lys(Me,) released from endocytosed labeled glycoprotein, or more directly from [1,2,3,4-'4C]y-butyrobetaine, coincided 'Note Added in Proof-Since submission of this manuscript, a paper has appeared (Thoma, W.J., and Henderson, L. M. (1984) Biochim. Biophys. Acta 797, ) that in essence removes this area of disagreement concerning the effect of ascorbate deficiency on liver carnitine content and on the levels of y-butyrobetaine hydroxylase activity. The reported studies, coming from the same laboratory as those of Nelson et al. (23), now show that liver carnitine levels were indeed significantly depressed in scorbutic guinea pigs, as was the in vivo conversion of [methyl-"c]y-butyrobetaine to ["Clcarnitine. Carnitine on Biosynthesis with hepatic accumulation of y-butyrobetaine. Results with the isolated guinea pig livers perfused simultaneously with [methyl-3h]lys(me3)-asialofetuin and [1,2,3,4-14C]y-buty- robetaine are therefore unambiguous, internally consistent, and unequivocally support the conclusion that liver y-butyrobetaine hydroxylase but not Lys(Me3) hydroxylase becomes rate-limiting for carnitine synthesis by the ascorbate-deficient animal. 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