Main

The EFA, namely, LA and ALA, were discovered in 1930 (1). Since that time, much has been learned about the biochemistry and physiologic functions of the EFA, although recent studies have raised important questions about the role of linoleic acid in EFA deficiency (2). Each EFA is the parent member of a family of fatty acids that have different biologic functions. For example, LA prevents water loss through the skin (3), arachidonic acid is the precursor of all of the main eicosanoids, EPA and DHA are effective in reducing plasma triacylglycerol levels (4), and DHA plays an important role in excitable tissues such as the brain, retina, and heart (5–7).

There has been much discussion in the literature recently on the role(s) of DHA in the nervous system (8) and on how to provide optimal neural DHA levels during the period of brain growth. Discussion has centered on the importance of dietary DHA compared with dietary ALA, because it is recognized that the conversion of ALA to DHA is limited in infants and adults (9–11). The relevance in infants is that human milk contains DHA, whereas many infant formulas do not. The question of whether formulas with ALA can provide sufficient neural DHA therefore is under discussion (11, 12). In guinea pigs, dietary ALA is about one-tenth as effective as dietary DHA as a dietary source for retinal and brain DHA levels (13). Diversion of ALA to other catabolic or anabolic pathways might explain the ineffectiveness of ALA as a source of tissue DHA. One such pathway is β-oxidation, whereby ALA is oxidized rapidly compared with other PUFA (14). This route could account for the substantial loss of ALA from the body, because diets rich in LA (LA:ALA >4:1) are associated with nearly double the rates of β-oxidation of ALA than are diets containing low levels of LA (LA:ALA at 0.5:1) (15).

The purpose of our study was to search for other pathways of ALA metabolism that could account for losses of ALA from the body. To do this, we followed the fate of a single dose of 14C-labeled ALA into all tissue lipids in the guinea pig.

MATERIALS AND METHODS

Four weanling (3-wk-old) male guinea pigs (pigmented strain) were maintained for 21 d on a semi-synthetic diet (6) containing 10% fat, which was a blend of six common vegetable oils. The fatty acid composition of this blend was 11% lauric plus myristic acid, 28% palmitic acid, 4% stearic acid, 35% oleic acid, 18.6% LA, and 2.8% ALA. We have previously raised guinea pigs for up to 16 wk from weaning on similar diets (6). After 19 d on the diet, each guinea pig was fed an oral dose of 4 μCi of 14C-labeled ALA (9,12,15[14C]linolenic acid) (NEN Life Sciences Product Inc., Boston, MA), specific activity 1.9240 GBq/mmol, mixed in 0.4 mL of olive oil, and then the guinea pigs were returned to their cages for 48 h, where they had normal access to food and water. Next, the guinea pigs were asphyxiated in CO2 gas, the head was severed from the body, and the skin and fur were removed from the skull and carcass of the body, respectively. The other tissues were collected, washed free from blood in ice-cold normal saline, and dried with blotting paper; then the total weight of the organs was recorded, and the tissues were stored at −20°C. All procedures conducted on the animals were approved by our Institutional Animal Ethics Committee.

Lipids were extracted from approximately 2 g of each tissue with chloroform-methanol, as described previously (13). One aliquot of each tissue lipid was counted in a scintillation counter, another aliquot was converted to fatty acid methyl esters for determination of the tissue fatty acid content by capillary GLC using C23:0 as an internal standard, and a final aliquot was subjected to TLC on silica gel G to separate the neutral lipids into fractions (13), which were then scraped into vials and counted in the scintillation counter. Some fatty acid methyl esters from different tissues were examined by silver-nitrate TLC to separate trienoic from dienoic and tetraenoic fractions with petroleum ether/diethyl ether (40/60, vol/vol) (16).

RESULTS

The weight gain attained by the four animals was 114 ± 18 g in the 3 wk of the experiment (from 408 g at the start to 522 g at the finish). Most of the ALA (mass) was deposited in carcass (muscle plus bone), skin and fur (of the carcass), and adipose tissue, in approximately equal proportions (Table 1). The carcass was the main site of deposition of long-chain n-3 PUFA. EPA was also found in substantial proportions in the skin and fur (carcass), and DHA in the brain, skin, and fur (carcass). LA was the major PUFA in the whole body (11.63 g), followed by ALA (1.54 g), arachidonic acid (0.52 g), and relatively small amounts of long-chain omega-3 PUFA (0.17 g). The carcass was the major reservoir of PUFA throughout the body.

Table 1 The individual n-3 PUFA content of tissues and incorporation of 14C-labeled LA in the guinea pig* * Results are shown as mean ± SD. ND = not determined. † The fatty acid contents (mg/tissue) were determined by capillary gas-liquid chromatography using an internal standard of C23:0. ‡ Results are expressed as the percent recovery of the dose of 4 μCi 14C-labeled ALA (8.88 × 106 dpm) which was given orally to each animal (n = 4) 48 h before it was killed.
Full size table

The distribution of 14C derived from the labeled ALA throughout the body lipids revealed that 46% of the label was recovered in the skin and fur (carcass plus head), 6% in the carcass (muscle plus bone), 4% in adipose, 3% in liver, and less than 0.1% in the brain, with only 61% of the administered label being recovered in the body lipids, as shown in Table 1. Whereas the tissues were washed in saline to remove blood, we cannot exclude the possibility that residual blood could have been present in some tissues. No blood sample was taken for counting in this study; however, it has been reported that 14C-LA disappears within several hours from the blood into tissues in guinea pigs after intravenous dosing (17). When the data were expressed as a specific activity (dpm/g ALA in each tissue), the skin and fur (head) had the highest value, followed by the liver, then all other tissues, which had about 1/10–1/20th activity of skin and fur (head). The specific activity in skin and fur (head) was more than 16 times that of the skin and fur (carcass). Tissue lipids were saponified and methylated to form fatty acid methyl esters and cholesterol, and most of the 14C label in the highly labeled tissues (adipose tissue, liver, skin, and fur) was in fatty acids of the lipids (determined as methyl esters, >92%), the balance being found in tissue cholesterol. Separation of these fatty acid methyl esters by Ag+-ion TLC revealed that most 14C was found in the trienoic fraction (>70%). We routinely use this technique to separate fatty acid methyl esters with zero to six double bonds, and the solvent system chosen clearly separated methyl esters with three double bonds from those with two and four double bonds, based on the separation of standards run on the same TLC plate. In other words, the label was still in ALA or another fatty acid with three double bonds. Examination of skin and fur separately revealed that 54% of the 14C label was in lipids extracted from the fur and 46% in lipids extracted from the skin. In both the skin and fur, more than 70% of the 14C label was found in the FFA fraction, the rest being in cholesterol and polar lipids. In contrast, in liver, adipose and carcass lipids, the 14C label was mainly in the triacylglycerol and polar lipid fractions.

DISCUSSION

The aim of this study was to search for reasons why ALA is a poor substrate for DHA synthesis. To do this, we determined the distribution of ALA (mg/tissue) and then the fate of 14C-labeled ALA throughout the body in young guinea pigs. The major sites for ALA accumulation (mg ALA/tissue) were the carcass, skin, fur, and adipose tissue, these tissues accounting for more than 90% of the whole body ALA. Skin has not previously been reported to be a major site of ALA deposition.

We found that less ALA (relative to LA) was recovered in the whole body lipids compared with that present in the diet. The ratio of LA:ALA in the diet was 6.64:1, and during the experiment, LA and ALA were deposited in the body, but not in this ratio. The whole body LA:ALA was 7.61 ± 0.39 (mean ± SD), resulting from a whole body content of LA of 11635 ± 644 mg (mean ± SD) and a whole body content of ALA of 1529 ± 151 mg. The difference between the diet and whole body LA:ALA suggests that relative to LA, some of the dietary ALA disappeared, perhaps during digestion and absorption, through β-oxidation, metabolism to long-chain n-3 PUFA, or via another route.

Only 61% of the label was recovered in whole body tissue lipids, which suggests that 39% of the 14C-ALA given was lost in CO2 (β-oxidation) or in feces (not absorbed). This proportion of loss through β-oxidation is of the same order of magnitude as that found in rats in two studies, wherein approximately 40–50% of the dose of radiolabeled ALA was found in expired CO2 over a 24-h period (14, 15).

The skin plus fur was the most labeled of all tissues examined. The specific activity (dpm/g ALA) of the different tissues revealed that the skin and fur of the head was four times more labeled than the liver, which in turn was more than twice as active as most other tissues. Carcass had the most mass of ALA, but it was not the most highly labeled tissue. When the fur was pulled from the skin of the carcass and head, it was found that 54% of the label was in the fur. Because it was not possible to observe any lipid mass in the extracted fur on the TLC plate (the fractions collected were identified by means of standards), the specific activity of the fur on its own must have been very high. New samples of fur were then cut from the skin with scissors, and after lipid extraction, more than 70% of the label in the fur was found in the FFA fraction, in a fatty acid with three double bonds (e.g. ALA). Because we found the 14C activity in the fur in three randomly selected sites in each of four animals at similar dpm/g fur on each occasion, we do not believe that it is likely that the fur was contaminated with label from the oral dosing 48 h earlier. Additional studies in guinea pigs (n = 8) that received orally administered 14C-LA did not find the same extent of label in the fur (Fu and Sinclair, unpublished data), which supports our belief that the 14C-labeled ALA in the fur is a real finding and not the result of contamination. Another source of potential contamination could be from feces; however, there was no evidence of the fur on any of the animals being contaminated in this way. The finding of a high level of labeling in the fur in the FFA fraction with three double bonds does not exclude the possibility that the ALA might have been converted to longer chain PUFA in other tissues, such as liver and brain. This possibility was not investigated in this experiment. However, other studies in the guinea pig have suggested that there is a relatively slow conversion of ALA to DHA in tissues such as liver and heart, but perhaps not in the brain (13).

The high proportion of label in the skin and fur was unexpected, inasmuch as others who examined the fate of labeled ALA by whole-body autoradiography in the rat found that the most highly labeled tissues were liver, brown fat, and adrenal cortex. However, in that study, they did not report any data for skin or fur (18). It is possible that in that study the skin and fur were removed before autoradiography was begun. As a result of the unexpected finding of 14C-labeled ALA in skin and fur, we conducted additional studies in guinea pigs fed diets with different LA:ALA ratios and administered either 14C-LA or 14C -ALA. The most highly labeled tissue in the 14C-ALA experiment (n = 12 guinea pigs) was the skin and fur of the head, whereas in the 14C-LA experiment (n = 8), there was a different pattern of tissue labeling, with the specific activity being highest in the liver, then the brain > lung and spleen > heart > skin and fur (head) > skin and fur (carcass) (Fu and Sinclair, unpublished data). These data reveal that ALA and LA are not metabolized in a similar manner via this route.

It has been widely reported that LA plays an important role in skin in preventing water loss and in maintaining epidermal integrity (3, 19). However, some studies have indicated that ALA might also play a role in skin and fur function. For example, an early study in rats showed that linseed oil contained a factor that promoted fur growth, compared with a fat-deficient control group, and that ALA was more effective than LA in restoring fur growth (20). A later study in capuchin monkeys reported skin lesions, fur loss, and abnormal behavior on a high-LA, low-ALA diet, and that there was a restoration of normal skin and fur appearance subsequent to the inclusion of linseed oil into the diet (21). In terms of water loss through the skin, it is well known that LA prevents abnormal water loss, which occurs in EFA deficiency; however, in these studies, ALA was ineffective (3). A case report of three elderly subjects revealed that low ALA intake was associated with dry, scaly, and atrophic skin (22), although there was no measurement of water loss in these subjects. In rhesus monkeys, ALA deficiency is associated with increased water intake; however, this was balanced by increased urination, rather than water loss through the skin (23).

Based on the results obtained in this experiment, it is possible to speculate that in the guinea pig, ALA may have a function in relation to fur, perhaps as a secreted lipid from sebaceous glands to protect the fur from damage by water, light, or other agents. What is the relevance of this work to human infants? If there is substantial disposal of ALA via sebaceous glands in humans, it might account for why ALA rarely accumulates in most tissues (24, 25) and why ALA is an inefficient substrate for DHA synthesis. Furthermore, if there is deposition of ALA in the skin, it might play a role in the prevention of the colonization of the skin by Staphylococcus aureus (26). Finally, the identification of substantial quantities of ALA, EPA, DPA, and DHA in the carcass and skin suggests that these could be potentially important reservoirs of omega-3 PUFA in the body. The importance of these issues in humans and other species remains to be confirmed.