Retinol and retinyl ester concentrations in rat tissues during total parenteral nutrition

Research Communications Retinol and retinyl ester concentrations in rat tissues during total parenteral nutrition Anne Lespine, Brigitte Periquet, Jesus Garcia, Jacques Ghisolfi, and Jean-Paul Thouvenot Groupe d’Etudes en Nutrition Infantile, Hoˆpital Purpan, Toulouse, Cedex, France

We have previously observed that continuous total parenteral nutrition (TPN) that supplies retinyl palmitate induces a strong decrease of the circulating retinol, which is associated with an impaired hepatic production of retinol-binding protein. We have investigated the effect of 7 days of TPN on the retinol and retinyl ester concentrations in rat tissues, relative to the vitamin A status [n 5 30 for vitamin A-sufficient (A1) and n 5 30 for deficient (A2)]. Rats were cannulated for intravenous feeding (n 5 12 for TPN-A1, n 5 12 for TPN-A2) and were compared with their per os pair-fed counterparts (n 5 12 for O-A1 and n 5 12 for O-A2). Retinol and retinoic acid in serum and retinol and retinyl ester concentrations in liver, kidney, lung, heart, and testis were measured by high performance liquid chromatography. TPN induced a dramatic decrease in circulating retinol of A1 rats, whereas retinoic acid concentration in serum was unchanged. When TPN was given to A2 rats, retinol concentration in serum remained low. Lower retinol and retinyl ester concentrations were measured in the livers of TPN rats compared with orally pair-fed rats, no matter the initial vitamin A status (P , 0.02 for A1 and P , 0.01 for A2). By contrast, in extrahepatic tissues, retinol and retinyl ester concentrations were similar in TPN rats compared with orally pair-fed rats. Our results indicate that TPN induced a decrease of retinol in serum and retinyl esters in liver. However, TPN was able to maintain retinol and retinyl ester concentrations in extrahepatic tissues of vitamin A-sufficient rats and to restore retinol and retinyl ester concentrations in extrahepatic tissues of vitamin A-deficient rats, albeit a low circulating retinol. (J. Nutr. Biochem. 9:316 –323, 1998) © Elsevier Science Inc. 1998

Keywords: Vitamin A; total parenteral nutrition; rats

Introduction Vitamin A is involved in many functions such as vision, development, growth, and cellular differentiation.1 Vitamin A is a generic term that includes not only retinol, the major circulating form, and retinyl esters, the storing form, but also the active forms such as retinoic acid and retinaldehyde. The importance of providing an adequate amount of

Address correspondence and reprint requests to Dr. Anne Lespine, Laboratoire de Biochimie, Hoˆpital Purpan, 31059 Toulouse, Cedex, France. Received October 22, 1997; revised February 27, 1998; accepted March 10, 1998.

J. Nutr. Biochem. 9:316 –323, 1998 © Elsevier Science Inc. 1998 655 Avenue of the Americas, New York, NY 10010

this vitamin, whatever the route of administration, is now well recognized. Vitamin A from per os diet is delivered to the liver mainly as retinyl palmitate combined with dietary lipids in chylomicrons (for a review see Blomhoff et al.2). Total parenteral nutrition (TPN) is an intravenous nutritional supply used in cases of severe malnutrition or in patients who cannot tolerate oral feeding, such as in intestinal or pancreatic pathologies associated with malabsorption. In these instances, TPN becomes the only way to provide protein-caloric requirement. Nutritive solutions also contain lipids, vitamins, micronutrients, and minerals. Little is known about the fate of these compounds when they are directly perfused. Metabolic problems such as cholestasis,3 liver dysfunction,4 bowel resection, and intravascular lipid

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Vitamin A tissue distribution during TPN: Lespine et al. Table 1 Composition of the semisynthetic solid diets and the liquid nutritive solution Semisynthetic solid diet (g/100 g)

Energy (kJ/100 g) Glucide Dextrose Premix G4L1 Cellulose Amidon Protide (casein)2 Lipids3 Vitamin A: retinyl palmitate Vitamin mix4 Trace elements (mineral mix 1 oligoelements)5

A1

A2

333.0

333.0

50.0 2.8 2.5 15.2 20.0 5.0 1023 1.0

50.0 2.8 2.5 15.2 20.0 5.0 0 1.0

3.5

3.5

Liquid diet6 (mL/100 mL) 114.0 Glucose 50%

35.8

Hyperamin 30% Endolipid 20%7 Vitalipid8 Soluvit Mineral sol.9 Nonan

36.2 7.9 2.4 1.2 13.6 2.9

Before beginning the experiment, rats were fed per os during 4 weeks with the standard semisynthetic solid diet (A1 or A2). Perfused rats (TPN-A) received the nutritive solution during 7 days (228 mL/kg per rat per day), while pair-fed controls (O-A) received orally a solid semisynthetic diet containing retinyl palmitate supplying the same amount of energy and nutrients (80 g/kg per rat per day). 1 Premix G4L: cellulose 1 DL-methionin. 2 Casein: vitamin free and delipidated. 3 Lipids: In the standard semisynthetic solid diet lipids were a mixture of (g per 100 g of diet) glycerol 1, stearate 3.20, linoleate 0.72, linolenate 0.10. 4 Vitamin mix provided (mg per 100 g of diet): ergo calciferol 0.007, thyamin 2, riboflavine 1.5, pyridoxine 1, vitamin B7 15, cyanocobalamin 0.005, DL-a-tocopherol 17, vitamin K 4, nicotinamide 10, choline chloride 136, folate 0.5, biotine 0.030. 5 Mineral mixture 1 oligoelements (mg per 100 g of diet): phosphorus 770, sodium 400; KCl 600, MgSO4 100, FeSO4 30, CuSO4 12, ZnSO4 45, CoCl2 0.009. 6 All solutions used for the liquid diet preparation are listed in Material and methods. 7 Endolipid 20% (g per 100 mL of Endolipid): glycerol 2.5, soybean oil 20, egg lecithin 1.2. 8 Vitalipid infant (mg per 10 mL of Vitalipid): retinyl palmitate 1.25, D-L-a-tocopherol 6.4. 9 Mineral solution (mL per 100 mL of liquid diet): calcium gluconate 10% 4.46, NaCl 20% 2.2, KCl 10% 3.26, MgSO4 1.26, PO4NaK2 10%, 2.5 mL.

metabolism disturbance5 are often associated with TPN. The amount of vitamin A provided during TPN is usually based on the Recommended Dietary Allowance for oral vitamin A intake.6 However, several clinical studies report abnormalities of the vitamin A status associated with parenteral feeding. In adults undergoing long term TPN, vitamin A deficiency has been reported.7 An impaired distribution of vitamin A due to adsorption in the container or degradation with time has been suspected. However, the authors have also suggested an impaired metabolism of the vitamin A, as a result of the intravenous distribution. Very low birth infants are often born with limited liver reserves of retinyl esters.8 They also have a low plasma concentration of retinol and low circulating retinol-binding protein (RBP),9 the retinol specific transporter. Although an adequate amount of vitamin A is supplied by TPN to these preterm babies as soon as the first day of life, vitamin A status is not always normalized.10 –12 The route of administration may account for the impaired utilization of the vitamin A.11 Conversely, studies performed in rats subjected to experimental TPN have shown an hepatic accumulation of retinyl esters.13 Furthermore, in our laboratory low serum retinol and low retinol binding protein (RBP) were found in rats after TPN.14 All these observations revealed the necessity to better describe the vitamin A metabolism when provided intravenously. The aim of this study was to follow the tissue content of retinol and retinyl ester in an experimental model of rats submitted to TPN. Nutrients and micronutrients, including vitamin A, were provided to rats intravenously by continuous perfusion. In clinical settings, patients undergoing TPN

often have a low vitamin A status as a result of malabsorption or reduction of food intake. Therefore, the rats used in this study were vitamin A-sufficient (A1) or vitamin A-deficient (A2).

Methods and materials Animals and diet Weaning male Wistar 7-week-old rats from Janvier (Le GenestSt-Isle, France) were individually housed in a temperature and light-controlled room (22–25°C and 12-hour light-dark cycle). Over 4 weeks, rats were fed a standard semisynthetic solid diet (UAR Laboratory, Villemoisson-sur-Orge, France) containing 20 mmol/kg retinyl palmitate (A1 group) or less than 0.2 mmol/kg vitamin A (A2 group). The details of the standard semisynthetic solid diet composition are reported in Table 1. Vitamin A status was determined in a set of rats at the beginning of the experiment (A1, A2). At that time, A1 rats (n 5 28) weighed 239 6 3 g and A2 rats were significantly lighter at 205 6 5 g (n 5 28, P , 0.001).

Total parenteral nutrition For surgery, rats were anesthetized with ketamine 72.5 mg/kg and a silastic catheter (Sigma Medical, Nanterre, France) was inserted into the right jugular vein, exteriorized at the nape of the neck via a subcutaneous tunnel. The perfusion system allowed the animals to move in the metabolic cage. The rats were maintained 24 hours under continuous perfusion of saline 0.9% via a volumetric infusion pump, with access to vitamin A-deficient food. Starting flow rate was 1.0 mL/hr and was progressively increased to 2.4 mL/hr. From the second day, they were not allowed to drink or to

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Research Communications eat and they were perfused with the liquid TPN solution (228 mL/kg per day). Popp and Wagner15 had previously shown that a period of 4 days of TPN was needed for rats to recover from surgery. Therefore we performed TPN for 7 days. Additional experiments were performed in order to explore the effect of the experimental procedure on the vitamin A status of the rats. A total of eight vitamin A-sufficient rats were used for these studies. Two rats were surgically implanted with a catheter into the jugular vein. The catheter was tunneled subcutaneously and exteriorized at the nape of the neck. The rats received a continuous infusion of sterile saline 0.9% throughout the experiment (7 days), at a flow rate of 2.4 mL/hr. During this period the rats had access to water and food. In parallel, oral counterparts were pair-fed per os. Following this experiment, two other rats were operated, and the right jugular vein was ligated. The rats were sutured as performed for TPN or intravenous saline rats but no catheter was implanted in the jugular vein. As above, orally pair-fed rats without any surgery were studied (oral group). After experiments (7 days) animals were sacrificed by exsanguination. Blood, liver, and tissue samples were collected for vitamin A analysis.

Preparation of the TPN solution The TPN solution was freshly prepared every 48 hours under sterile conditions. The volumes of the solutions that were mixed for 100 mL of the TPN solution are reported in Table 1. Glucose 50%, hyperamin 30% as protein source, and endolipid 20% came from Bruneau (Paris, France); trace elements and vitamins were supplied by Nonan (Aguettant, Lyon, France); and Vitalipid Infant and Soluvit were from Kabi Vitrum (Stockholm, Sweden). Total energy intake was 1170 kjoul/kg per rat per day. The energy distribution was adapted to the rat standard solid diet with a glucose/protein/lipid ratio of 65/23/12. Endolipid 20% was a lipid emulsion made of soybean oil (20%) emulsified with egg lysophospholipid (1%). Vitalipid Infant provided the liposoluble vitamins (retinyl palmitate and D-L-a-tocopherol; Table 1) combined with a 10% lipid emulsion. The TPN rats received 1.29 mmol retinyl palmitate/kg per rat per day. In parallel, pair-fed control rats (O-A2 and O-A1) received per os the standard semisynthetic solid diet containing vitamin A. The maximal efficiency of intestinal absorption for vitamin A in the rat, which was reported to be 80%,16,17 was taken into account and control rats received per os 1.52 mmol retinyl palmitate/kg per rat per day. The pair-feeding was performed in order to provide to per os fed rats a similar amount of energy and vitamin A as the TPN pairwise animal. Therefore, the volume of the nutritive solution administered to TPN fed rats was noted every day. The pair-fed per os counterpart received an amount of solid diet that corresponded to the amount of energy and vitamin A taken the day before by the TPN pairwise rat. We have also checked the liposoluble vitamin stability in our perfusion system by measuring vitamins A and E in the nutritive solutions. All the vitamin A was recovered intact after one passage through the tubing. Delivery was 100% and 85% of the initial concentration, after 24 hours and 48 hours, respectively, when standing at room temperature and light exposure. From these results we ruled out any possibility of adsorption of vitamin A into the infusion system or any degradation with light or time using vitamin A combined with lipids.

Blood and tissue collection For TPN rats, the perfusion was discontinued, whereas for per os rats, food was withheld 12 hours before sacrifice. The rats were anesthetized and exsanguinated by the abdominal aorta. The serum was obtained after 15 minutes of centrifugation at 2000 3 g. Liver, kidney, lung, heart, and testis tissues were collected, and rinsed in ice-cold NaCl 0.9% solution. After blotting the excess saline with

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filter paper, each tissue sample was weighed and the whole organ was homogenized for vitamin A analysis.

Vitamin A extraction and analysis Extraction and analysis of retinol and retinyl esters from the serum and the tissues were performed according to Periquet et al.18 Briefly, the whole liver, kidney, lung, heart, or testis was homogenized with a potter-Elvehjem homogenizer in 9 volumes of buffer (0.25 mol/L sucrose; 0.05 mol/L Tris/HCl, pH 7.6; 0.025 mol/L Kcl; 0.005 mol/L MgCl2). Serum or tissue homogenate was mixed with one volume (v/v) of ethanol solution containing 10 mg/L retinyl acetate used as internal standard. Samples were extracted with 5 volumes of n-hexane. After centrifugation, 500 mL of the upper phase were evaporated under nitrogen stream in subdued light, dissolved in methanol, and injected into a high performance liquid chromatography (HPLC) system (Philips Model 4100 liquid chromatography). Column was a Spherisorb ODS 2, 5 mm, 25 cm (Chromato-Sud, Bordeaux, France). Chromatography was performed at 50°C with methanol as solvent. The detecting wavelength was 325 nm. This system allowed detection of seven different retinyl esters. Retinyl myristate/retinyl palmitoleate and retinyl palmitate/retinyl oleate were not separated in our conditions, due to opposing effects of increasing unsaturation and increasing chain length.19 Retinyl oleate was considered as a minor form in the tissues.20 Measurement of retinoic acid was performed according to Periquet et al.,21 but was modified slightly. Briefly, 3 mL of serum were extracted with hexane in presence of 2 N KOH, after the addition of internal standard (Acitretin, gift from Dr. Klauss, Hoffman-La Roche, Basel, Switzerland). The aqueous phase was then acidified by adding 2N HCL and extracted with hexane. The organic phase was run on aminopropyl column to remove the neutral lipid. Retinoic acid bound to the column was eluted with diethyl ether, acetic acid (97/3, v/v), evaporated under nitrogen stream, and dissolved in the mobile phase (acetonitrile/ water/acetic acid, 80/19.8/0.2, v/v/v). The sample was analysed on a HPLC column of Spherisorb ODS 2, 5 mm, 4.6 mm by 10 cm (Chromato-Sud, Bordeaux, France). The detecting wavelength was 350 nm.

Animal health and organ weight The biological variables of the TPN rats were within normal range as previously reported.14 During TPN, A2 and A1 rat body weight increased similarly with a mean rate of 3 g/day. Urine was collected every day during perfusion and analysed for proteins, glucose, ketone bodies, blood, and pH with Ames reagent Strips (Miles Laboratory, USA). These variables were maintained at normal levels during the experimental procedure. The liver, lung, kidney, and heart weights were unaffected by vitamin A deficiency and by TPN over time. However, an increase over time was noticed in the testes weight of A1 rats. Such an increase was not observed in A2.

Vitamin A status of A1 and A2 rats After 4 weeks of vitamin A supplying diet (A1 rats), the circulating retinol was 1.50 6 0.17 mmol/L, hepatic stores of retinyl ester being 459.0 6 11.2 nmol/g liver. From previous experiments, we have observed that A2 rats had a hepatic retinyl ester concentration ranging between 0 and 28.6 nmol/g liver. Two distinctly deficient populations were defined: (1) animals with normal serum retinol (approximately 1.75 mmol/L) and (2) animals with low serum retinol (approximately 0.70 mmol/L). The first group had liver retinyl ester concentrations over 9.5 nmol/g liver. We have used here the second population, which had hepatic retinyl esters below 9.5 nmol/g, which correlated with serum

Vitamin A tissue distribution during TPN: Lespine et al. Table 2 Effect of total parenteral nutrition on vitamin A concentration in the rat liver Hepatic vitamin A (nmol/g)

Vitamin A-sufficient (A1) Vitamin A-deficient (A2)

Oral

TPN

P-value

% decrease of liver vitamin A after TPN

607.6 6 69.9 (n 5 9) 77.1 6 9.2 (n 5 11)

476.6 6 45.3 (n 5 9) 52.9 6 9.8 (n 5 11)

0.015 0.008

18.6 6 5.1% 31.2 6 7.2%

Experiments were performed in A1 rats (n 5 18) or in A2 rats (n 5 22). The rats were submitted to TPN during 7 days and compared with their pair-fed counterparts receiving per os a semisynthetic diet (oral). Vitamin A (retinol 1 retinyl esters) was measured in the whole liver. Pairwise animals were compared using a Wilcoxon signed ranks test. Data are means 6 SEM. The percent of decrease of vitamin A in TPN rat liver was calculated for pairwise animals (100% correspond to retinyl esters 1 retinol in the liver of orally fed animals).

retinol (n 5 6, r 5 0.82, P , 0.007). Those animals were considered as a reflection of the outset of the vitamin A deficiency. These observations are in agreement with a previous work performed in our laboratory.19 In order to select A2 rats with low serum retinol concentration, blood was withdrawn by the tail vein the day before the experiment, and retinol concentration was determined by 100 mL of serum.

Statistical analysis The Bartlett test was used to test homogeneity of variance of the two starting groups (A2 and A1). If variances were homogeneous, the mean differences were compared by Student’s t-test. Differences between the six groups (A2, A1, TPN-A2, TPN-A1, O-A2, and O-A1) and changes within groups over time were evaluated by analysis of variance (one-way ANOVA). If ANOVA indicated statistically significant differences (P , 0.05), mean values were ranked by the Tukey’s test. For comparison of pairwise animals, a Wilkinson signed ranks test was used. Statistical analysis was conducted using the SYSTAT statistical software.21

Results Effect of TPN on retinol and retinoic acid concentrations in rat serum Retinol was decreased in rat serum during TPN to 0.63 6 0.01 mmol/L in TPN-A1 rats (P , 0.001 when compared with A1 rats). In the TPN-A2 serum, retinol was maintained at a low concentration (0.66 6 0.01 mmol/L). By contrast, when vitamin A was provided per os to A2 rats, the serum retinol was normalized (1.36 6 0.01 mmol/L). The concentration of retinoic acid was determined in the serum of five O-A1 and five TPN-A1 animals. The technique allowed us to separate three distinct isomers in control rat serum, with the following concentrations: all-trans retinoic acid (5.55 6 1.30 nmol/L); 13-cis retinoic acid (2.55 6 0.31 nmol/L); and 9-cis retinoic acid: 2.66 6 0.86 nmol/L. After TPN none of the three isomer concentrations were changed (all-trans, 5.40 6 0.66; 13-cis, 2.31 6 0.76, and 9-cis, 2.70 6 0.33 mmol/L, mean 6 SEM of five animals).

Effect of TPN on the liver retinol and retinyl ester concentration We then explored the liver vitamin A content relative to the low serum level observed during TPN. In vitamin A sufficient rats, a reproducible and significant lower liver retinyl ester concentration was observed after 7 days of TPN compared with the per os pair-fed O-A1 counterparts. Thus,

after 7 days of experiment, in TPN-A1 rats the amounts of retinyl ester and retinol were 464.2 6 42.5 and 12.4 6 4.6 nmol/g liver, respectively, whereas in O-A1 the reserves were 590.8 6 66.3 nmol/g retinyl esters and retinol was 16.8 6 6.6 nmol/g (Table 2; P , 0.015 for retinyl ester 1 retinol concentration). Taking into account the retinyl ester concentration in A1 animals before the experiment (459.0 6 11.0 nmol/g), this indicated that the increase of retinyl ester stores over time, observed in the whole liver of O-A1 rats, were inhibited by TPN. Whatever the route of administration, in the A2 rats vitamin A supplied during 7 days induced an increase of the hepatic retinol and retinyl ester content. However, in 7 days TPN-A2 rats, the hepatic retinyl esters were significantly lower when compared with their per os pair-fed O-A2 counterparts (Table 2, P , 0.008). The repletion with time of retinyl esters in the liver was slow in TPN-A2 compared with O-A2. This resulted in a higher retinyl ester content in O-A2 compared with TPN-A2 (82.8 6 11.2 nmol/g and 54.8 6 8.3 nmol/g tissue, respectively). In the A1 group, the major molecular species of vitamin A were retinyl esters, only 1.2% to 2.9% of which were retinol (Table 3). Among retinyl esters, 78% to 85% was retinyl palmitate and 12% to 15% was retinyl stearate. The minor forms were retinyl laurate, myristate/palmitoleate, and linoleate, which accounted for less than 3%. Retinyl heptadecanoate and pentadecanoate contribution was 0.4% for each form in A1 animals and 0.2% in supplemented A2 animals (data not reported). This distribution was unchanged after TPN, except for retinyl linoleate. This minor form contributed to 0.4 6 0.0% of total retinyl esters in O-A1 and was increased to 1.9% in TPN-A1 (P , 0.02; Table 3). This retinyl ester distribution was identical in TPN-A2 and O-A2.

Effect of TPN on retinol and retinyl ester concentration in extrahepatic tissues In the A1 kidney, 93% of the vitamin A was present as retinol (4.6 6 0.3 nmol/g; Figure 1A). Only 0.20 6 0.02 nmol/g of retinyl ester was detected as retinyl palmitate. After 5 days of experiment, this distribution was not significantly changed. However, retinyl stearate, undetected in A1 rats, appeared in and contributed to 10% and 12% of total retinyl esters in O-A1 and TPN-A1, respectively. Retinyl myristate/palmitoleate appeared only in some TPN-A1 kidney (4.8 6 3.6%). In the A2 kidney, retinol J. Nutr. Biochem., 1998, vol. 9, June

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Research Communications Table 3 Retinol and retinyl ester distribution in rat liver during total parenteral nutrition (TPN) Percent of total vitamin A Groups

n

Retinol

Retinyl laurate

Retinyl myristate

Retinyl linoleate

Retinyl palmitate

Retinyl stearate

A1 TPN-A1 O-A1 A2 TPN-A2 O-A2

6 11 12 6 12 12

1.2 6 0.1 2.9 6 0.9 2.9 6 1.1 15.5 6 5.5 6.5 6 0.6 5.9 6 0.7

0.4 6 0.0 0.5 6 0.1 0.5 6 0.1 0 0.2 6 0.1 0.5 6 0.2

1.7 6 0.1 1.3 6 0.1 1.6 6 0.1 0 0.7 6 0.1 1.7 6 0.2

0.4 6 0.0 1.9 6 0.6a 0.6 6 0.1 0 0.8 6 0.2 0.3 6 0.1

83.2 6 1.1 77.7 6 2.1 78.7 6 2.1 84.4 85.2 6 2.8 84.8 6 1.8

12.3 6 0.9 14.9 6 5.2 14.8 6 1.8 0 12.9 6 2.7 12.7 6 1.4

Retinol and retinyl esters were measured in the whole liver homogenates in a set of vitamin A-sufficient rats (A1) and deficient rats (A2), representing vitamin A status before the experiment. After 7 days of TPN or oral feeding, vitamin A distribution was determined in the livers of TPN-A1, TPN-A2, O-A1, and O-A2 animals. The distribution of retinol and different retinyl ester was expressed as percent of total vitamin A. Data are means 6 SEM. 1P , 0.05 compared with A1.

was decreased by one third compared with A1 (P , 0.03), whereas an accumulation of retinyl esters was noted (3.4 6 1.3 nmol/g; P , 0.001 when compared with A1; Figure 1B). Whatever the route of vitamin A supply, retinyl esters remained high with 3.5 6 0.6 nmol/g in O-A2 and 2.8 6 0.9 nmol/g in TPN-A2. In parallel, retinol was increased (6.3 6 0.7 nmol/g for O-A2, P , 0.05; 4.2 6 0.7 nmol/g for TPN-A2, P 5 NS, versus 2.3 6 0.6 nmol/g in A2). An equivalent contribution of retinol and retinyl esters was then

observed, which constituted a major difference from what had been measured in the A1 experiment. In both A2supplemented groups (TPN-A2 and O-A2), a change in retinyl ester distribution was noticed, with a lower contribution of retinyl palmitate (approximately 50%), favoring the retinyl stearate. An equal participation of both species was then observed. In some cases, the appearance of retinyl myristate/palmitoleate was measured, accounting for 0.2 to 1.1% of total retinyl esters. Moreover a substantial amount

Figure 1 Effect of TPN on retinol and retinyl ester concentrations in extrahepatic tissues. Retinol, retinyl esters were measured in the whole tissue homogenate in a set of vitamin A-sufficient rats (A1) and deficient rats (A2), representing vitamin A status before the experiment. The A1 rats (2A) or the A2 rats (2B) were perfused for 7 days with the liquid diet or fed per os with the standard semisynthetic solid diet, containing retinyl palmitate. Tissue was homogenized and retinol and retinyl ester contents were determined by high performance liquid chromatography in the lung, kidney, heart, or testes. Data were as nmol/g 6 SEM of wet tissues. a, P , 0.05, and b, P , 0.001 when compared with A1.

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Vitamin A tissue distribution during TPN: Lespine et al. of retinyl linoleate was observed only in TPN-A2 (4.5 6 1.4%). Among the extrahepatic tissues studied, the A1 lungs had the higher tissue concentration of vitamin A; 90% were retinyl ester (18.1 6 1.5 nmol/g), retinol representing only 10% (1.9 6 0.4 nmol/g, Figure 1A). Among retinyl ester, the main species were retinyl palmitate (51.3 6 0.4%) and retinyl stearate (44.7 6 1.0%). The retinol and retinyl ester content and distribution did not vary after TPN except for the contribution of retinyl linoleate, which was increased (3.5 6 0.6% in TPN-A1 vs. 1.6 6 0.2 in A1; P , 0.002). In the A2 lung, retinol and retinyl ester content was depleted compared with A1. After vitamin A supply, retinyl esters were significantly restored and reached 5.6 6 1.2 nmol/g for TPN-A2 and 6.0 6 0.9 nmol/g for O-A2. A similar increase was observed for retinol that returned to A1 value (Figure 1B). In parallel, the pattern of retinyl esters was maintained similar to the one observed in A1. In the A1 heart, 8.4 nmol/g of vitamin A (retinol 1 retinyl ester) was measured, equally shared between retinol and retinyl esters (Figure 1A). TPN induced a nonsignificant change in this balance favoring retinyl esters. Most of retinyl ester was retinyl palmitate (between 57% and 84%, the remaining being retinyl stearate). Vitamin A deficiency induced a dramatic decrease in heart retinyl esters and a total disappearance of retinol (Figure 1B). Vitamin A supply induced a restoration of both forms of vitamin A that returned to normal values (see A1), as did the retinyl ester pattern. Retinyl linoleate was not detected in the heart. In the A1 testes, no retinol was found and all the vitamin A measured under our conditions was in the form of retinyl palmitate (1.1 nmole/g). During the experiment, the retinol appeared and contributed to 30% of total vitamin A, the remaining being retinyl palmitate. In A2 testes, retinyl ester concentration was low compared with A1. After vitamin A supply, retinyl esters were significantly increased. In both groups studied (TPN-A2 and O-A2), free retinol appeared and reached a value corresponding to the concentration found in O-A1 and TPN-A1. Additional experiments were performed in order to check a possible effect of the experimental procedure on vitamin A status in the rats. When animals were given intravenous saline during 7 days, no decrease in hepatic vitamin A was noticed compared with pair-fed per os counterpart (548.6 vs. 540.9 nmol/g, respectively; one experiment representative of two). Likewise, 7 days after the surgery alone, hepatic retinyl esters and retinol were unchanged (546.3 nmol/g). Furthermore, in the extrahepatic tissues the retinyl ester and retinol content were not modified by any of these experimental procedures. These experiments clearly confirmed that the decrease of hepatic vitamin A observed in the TPN group was related to the intravenous administration of the nutritive solution.

Discussion We have previously shown that TPN induced a dramatic decrease in circulating retinol in an experimental model of rats submitted to TPN. This decrease has been associated with an impaired RBP synthesis.14 In order to determine if the low serum retinol transporter induced by TPN is

representative of a low vitamin A status, we explored the vitamin A content in hepatic and extrahepatic tissues of rats receiving TPN. Therefore we have compared TPN rats and per os rats receiving an equivalent amount of energy, nutrients, and micronutrients, adapted to the recommended oral standard diet for rats. The comparison between the two routes of feeding studied here will inform on the efficiency of TPN in supplying vitamin A to rat tissues. This work focuses on the major form of vitamin A present in the body. Retinol reflects the transportation to tissues and the immediately available form for oxidation and action. The retinyl esters represent the storage form. The retinoic acid is the oxidized derivative of retinol, which is one of the major active forms. An increase in retinoic acid concentration was reported to induce a decrease of serum retinol in animal models.23 In our study we measured retinoic acid in serum, and found the presence of all-trans, 13-cis and 9-cis retinoic acid in rat serum, which account in control animals for 0.34%, 0.10%, and 0.20%, respectively, of total circulating retinoids. After TPN no change in the three retinoic acid isomer concentrations was measured in the serum. However, our technique does not allow measurement of retinaldehyde or other important metabolites such as retinoyl glucuronides, which also might be changed by TPN. Moreover, unchanged retinoic acid concentrations in serum during TPN does not exclude a change in plasma retinoic acid turnover. In normal vitamin A status, the liver contains 95% of the total vitamin A as retinyl esters and constitutes the main body reserve. In our study, most of the retinyl palmitate ingested by O-A1 rats during 7 days of experiment is recovered in the liver (90%). In the A2 rats, vitamin A oral feeding restored hepatic reserves at a lower rate and only 67% of the vitamin A supplied was found in the liver. We have shown that after TPN, the liver retinyl ester reserves were significantly decreased in the A1 and A2 groups, compared with their per os pair-fed counterparts (O-A1 and O-A2). Our results ruled out any effect of surgical stress on hepatic retinyl ester concentration. Moreover, the decrease in retinol and retinyl ester in the liver occurred only in TPN rats and not in rats given intravenous saline. From these data we conclude that the decrease of hepatic retinyl ester and retinol observed in the liver after TPN was related to the administration of the nutritive solution and not due to the experimental procedure. These results indicate that the balance between vitamin A infused and delivered to the liver during TPN, and that which is mobilized, has been impaired by TPN. Controversial results have been reported in a previous study. In this case, an accumulation of retinyl esters was measured in the rat liver after TPN.13 Vitamin A was supplied in a preparation of multi-vitamin infusion (MVI) mixed with retinyl palmitate, and TPN was performed without addition of lipids to the perfused solution. However, it has been established that MVI is not an inadequate formula for vitamin A intravenous supply.6 Furthermore, TPN is now performed with lipids in order to minimize side effects due to high amount of glucose infused. In our study we have used a lipid-containing nutritive solution, retinyl palmitate being combined with lipid emulsions (Vitalipid Infant, KabiVitrum, Sweden). This formula has been shown to be more stable than a J. Nutr. Biochem., 1998, vol. 9, June

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Research Communications vitamin A aqueous emulsion.24,25 The effect of TPN on hepatic vitamin A observed might be related to the formula of the vitamin A perfused and to the associated nutrients. A decreasing effect of TPN on hepatic retinyl ester stores probably is not a key point when TPN is performed in patients who have a normal hepatic vitamin A content. However, for long-term TPN or for patients with low hepatic retinyl ester stores at the beginning of TPN, such as in severe intestinal absorption disorder or in newborn or preterm infants, a slowing down in the formation of vitamin A hepatic stores might induce an incorrect availability of vitamin A. Thus, the delay observed in the formation of hepatic reserves reflects that the formula must be improved. In order to verify that the retinyl palmitate perfusion allows an adequate supply of vitamin A to the peripheral tissues, retinol and retinyl esters have been measured in several major vitamin A target tissues. The kidney plays a key role influencing the release of retinol-RBP from liver into circulation.26 An auxiliary role of this organ also has been described during vitamin A deficiency and in 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD)-treated rats, which has been attributed to an increase of acyl coenzyme A:retinol acyl transferase activity.27 In our experimental condition of deficiency, the A2 kidney becomes the major site of vitamin A storage and of available retinol, whereas total vitamin A in the liver was almost depleted. However a complete depletion of the kidney vitamin A has been reported after deficiency.16 This discrepancy can be explained by the long period of vitamin A deprivation of the animals used, our rats being just at the beginning of the total liver depletion. Our results strengthen the idea that the kidney plays a sparing role in the vitamin A status in the early stage of deficiency. Moreover we have shown that TPN is able to provide vitamin A in kidney from A1 and A2 rats. Lung contains retinyl ester20,28,29 and metabolizes retinyl palmitate from chylomicron remnants into retinoic acid.30 Preterm children have at birth immature lungs and low concentrations of serum retinol and hepatic retinyl esters.8,9 The key role of vitamin A in the lung development31 and in reversing emphysema in a rat model of disease32 has been demonstrated. We confirm here that the lung from A1 rats contains high amount of retinol and retinyl esters and that TPN allows a steady state level or an increase of the lung vitamin A content. During TPN, a direct uptake of the lipid emulsion with vitamin A by the tissue may contribute to the vitamin A lung concentration. Vitamin A is indispensable for normal development and function of the testes, retinol being essential in spermatogenesis.33 Moreover retinol is the only source of retinoic acid in testes, because this tissue is unable to take up retinoic acid from plasma.34 We have shown here that TPN is an efficient way to provide vitamin A to the testes of growing rats. Little is known about the regulation of heart vitamin A content. Our data have shown that heart tissue is sensitive to vitamin A deficiency and that retinyl palmitate supplied per os or by TPN is able to restore vitamin A in A2 heart. Lipid added to the perfused nutritive solutions originates from soybean oil, which is rich in linoleate and a-linolenate. In our TPN rats, an increase of retinyl linoleate in the liver, 322

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kidney, and lung was observed and might reflect the enrichment of the diet with polyunsaturated fatty acids. Considering the reported effects of a diet rich in polyunsaturated fatty acids on vitamin A metabolism,35–37 we cannot rule out that the quality of the lipid perfused is involved in this decrease of the hepatic stores. On the other hand, the effect of a polyunsaturated fatty acid-rich diet in TPN is not seen here on extrahepatic vitamin A content, but may be observed after longer intravenous perfusion. Our results show that in vitamin A deficient tissues, TPN induced an increase in retinol and retinyl esters, even though serum retinol concentration was low. These observations are supported by a previous work showing a direct uptake of infused, emulsified retinyl heptadecanoate by extrahepatic tissues, which was favored by vitamin A deficiency.29 Another work performed in transthyretin-deficient mice showed that, although the level of circulating RBP and retinol (only 5% of normal level) was low, the vitamin A status in extrahepatic tissues remained normal.38 Furthermore, a significant uptake of retinyl palmitate by the bone marrow has been described in hepatectomized rats.39 A preferential uptake of perfused emulsions by the spleen also has been observed in rats compared with perfused chylomicrons.40 Further investigations in our experimental model of TPN, closely reproducing artificial nutrition used in the clinic, may add important information regarding the utilization and transport of vitamin A to hepatic and extrahepatic tissues during parenteral feeding. The data reported emphasize that, in addition to the liver being the main vitamin A reserve, peripheral tissues may have a relative independence toward vitamin A uptake and utilization.

Acknowledgments We are grateful to William S. Blaner, Xavier Collet, Franc¸oise Hulin, and Janice Au’Young for their helpful discussions.

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