well have provided sufficient protection against such imbalances. 1. Reeves JP. Accumulation of amino acids by lysosomes incubated with amino acid methyl esters. J Biol Chem 1979;254:8914-21 2. Milne MD, Crawford MA, Girao CB, Loughridge

LW. The metabolic disorder in Hartnup disease. Q J Med 1960;29:407-21 3. Scriver CR. Hartnup disease. N Engl J Med 1965; 273 1530-2 4. Jonas AJ, Butler IJ. Circumvention of defective neutral amino acid transport in Hartnup disease using tryptophan ethyl ester. J Clin Invest 1989;84:200-4

PHOSPHATIDYLCHOLINE, LYSOPHOSPHATIDYLCHOLINE, AND LIPOPROTEIN SECRETION BY CHOLINE-DEFICIENT HEPATOCYTES Choline deficiency causes impaired secretion of very-low-density lipoproteins by hepatocytes and leads to fat accumulation in liver. This process can be reversed by phosphatidylcholine biosynthesis, phosphatidylethanolamine methylation, and acylation of exogenous lysophosphatidylcholine.

Feeding rats a diet deficient in choline and low in methionine rapidly induces fatty liver, lipid peroxidation, DNA damage and repair, and liver cell proliferation, and ultimately results in liver Choline deficiency appears to act as both an initiator and promoter in liver cancer, and thus represents the best-documented case in which the absence of a single dietary constituent causes cancer. The rapidity of triglyceride accumulation (within 8 h), which is followed by lipid peroxidation, indicates that aberrant lipid metabolism may be an important early step of choline deficiency. Yao and Vance’O hypothesized that impaired secretion of verylow-density lipoprotein (YLDL) by liver may account for the induction of fatty liver and the reduction in plasma triglycerides and other lipids. They have shown that hepatocytes from rats fed a choline-deficient diet and cultured in a medium lacking choline have a reduced secretion of VLDL but not of high-density lipoprotein. The addition of choline or methionine stimulated synthesis of phosphatidylcholine (PC) and increased the secretion of PC and triglycerides by two- to threefold. The effect of methionine 24

NUTRITION REVlEWSlVOL 48, NO IIJANUARY 1990

was attributed to its stimulation of PC synthesis by methylation of phosphatidylethanolamine. Choline deficiency had no effect on [3H]leucine incorporation into cellular VLDL apolipoproteins; hence, they concluded that active biosynthesis of PC from choline (via the CDP-choline pathway) or methionine (via methylation of phosphatidylethanolamine) is required for VLDL secretion. Two other sources of choline are lysophosphatidylcholine (IysoPC) and sphingomyelin. LysoPC is typically a minor constituent of tissues, but it represents one of the major phospholipids of plasma. Its presence in blood (where it is bound to albumin) is often ascribed to the action of lecithin-cholesterol acyltransferase (LCAT), which catalyzes the transfer of a fatty acid from the 2-position of PC to cholesterol in the formation of cholesterol esters. However, this idea is undergoing reevaluation, and it appears that hepatic secretion contributes a significant amount of IysoPC. The evidence for this includes: 1) the observation that plasma still contains a considerable amount of IysoPC in patients with congenital defects in LCAT;” 2) studies

with perfused liver1* showing that IysoPC is produced and the amount found in a lipidfree perfusion medium containing albumin exceeds that of PC; and 3) cultured hepatocytes also release IysoPC, and unsaturated fatty acids stimulate its secretion (and appear in the l ~ s o P C ) . ’ ~Unlike - ~ ~ the products of the LCAT reaction, which are predominately saturated, the IysoPC secreted by liver has mainly unsaturated fatty acids. Taken together, these findings suggest that IysoPC is secreted by liver and may represent another pathway for delivering choline (and perhaps unsaturated fatty acids) to cells. Robinson et a1.16 found that IysoPC secretion continues in rat hepatocytes deficient in choline and methionine, in contrast to the defective synthesis and secretion of PC. Release of IysoPC was strongly dependent on albumin, with which most of the lySOPC was associated in the culture medium. The hepatocytes were also shown to take up IysoPC and convert it to PC. When choline-deficient hepatocytes were supplemented with IysoPC, the mass of PC increased by 30-35% in the first 4 h. LysoPC supplementation was as effective as choline in restoring the secretion of VLDL as judged by the secretion of triacylglycerol, PC, and apolipoproteins. This finding adds additional strength to the hypothesis of Yao and VancelO that PC formation is critical for VLDL secretion. Subsequent investigations have found that the choline headgroup moiety is specifically required for normal VLDL secretion; N,N-dimethylethanolamine is converted to the phosphatidyl analogue and normalizes triacylglycerol synthesis in choline-deficient hepatocytes in the same way as choline but does not restore VLDL ~ e c r e t i 0 n . l ~ These investigations have clarified the importance of choline-containing lipids in lipid metabolism and lipoprotein secretion by liver and appear to provide the mechanism for the lipid abnormalities of choline deficiency. To the extent that the fatty liver and lipid peroxidation may contribute to hepatocarcinogenesis, these findings may explain the initial events in this process as

well. Lastly, the emerging picture of IysoPC as an important secretory lipid warrants additional evaluation in the transport of choline equivalents and unsaturated fatty acids. 1. Copeland DH, Salmon WD. The occurrence of neoplasia in the liver, lungs, and other tissues of rats as a result of prolonged choline deficiency. Am J Pathol 1946;22:1059-79 2. Mookerjea S,Park CE, Kuksis A. Lipid profiles of plasma lipoproteins of fasted and fed normal and choline-deficient rats. Lipids 1975;10:374-82 3. Mikol YB, Hoover KL, Creasia D, Poirier LA. Hepatocarcinogenesis in rats fed methyl-deficient, amino acid defined diets. Carcinogenesis 1983;4:1619-29 4. Goshal AK, Farber E. The induction of liver cancer by a dietary deficiency of choline and methionine without added carcinogens. Carcinogenesis 1984;5:1367-70 5. Rushmore TH, Lim YP, Farber E, Ghoshal AK. Rapid lipid peroxidation in the nuclear fraction of rat liver induced by a diet deficient in choline and rnethionine. Cancer Letts 1984;24:251-5 6. Yokoyama S, Sells MA, Reddy TV, Lombardi 6. Hepatocarcinogenic and promoting action of a choline-devoid diet in the rat. Cancer Res 1985; 4512834-42 7. Rushmore TH, Farber E, Ghoshal AK, Parodi S, Pala M, Taningher M. A choline-devoid diet, carcinogenic in the rat, induces DNA damage and repair. Carcinogenesis 1986;7:1677-80 8. Ghoshal AK, Laconi E, Willernsen F, Ghoshal A, Rushmore TH, Farber E. Modulation of calcium by the carcinogenic process in the liver induced by a choline-deficient diet. Can J Physiol Pharmacol 1987;65:478-82 9. Chandar N, Lambardi B. Liver cell proliferation and incidence of hepatocellular carcinomas in rats fed consecutively a choline-devoid and a choline-supplemented diet. Carcinogenesis 1988;9:259-63 10. Yao 2 , Vance DE. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J Biol Chem 1988;263:2998-3004 11. Norum KR, Gjone E. Familial plasma lecithin: cholesterol acyltransferase deficiency. Scand J Clin Lab Invest 1967;20:231-43 12. Sekas G. Patton GM, Lincoln EC, Robins SJ. Origin of plasma lysophosphatidylcholine: evidence for direct hepatic secretion in the rat. J Lab Clin Med 1985;105:190-4 13. Mangiapane EH, Brindley DN. Effects of dexa-

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methasone and insulin on the synthesis of triacylglycerois and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes. Biochem J 1986;233:151-60 14. Graham A, Bennett AJ, McLean AAM, Zammit VA, Brindley DN. Factors regulating the secretion of lysophosphatidylcholine by rat hepatocytes compared with the synthesis and secretion of phosphatidylcholine and triacylglycerol. Biochem J 1988 ;253 1687-92 15. Baisted DJ, Robinson BS, Vance DE. Albumin

stimulates the release of lysophosphatidylcholine from cultivated rat hepatocytes. Biochem J 1988 ;253 :693- 701 16. Robinson BS, Yao Z, Baisted DJ, Vance DE. Lysophosphatidylcholine metabolism and lipoprotein secretion by cultured rat hepatocytes deficient in choline. Biochem J 1989;260:207-14 17. Yao Z. Vance DE. Head group specificity i n the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes. J Biol Chem 1989;264: 11373-80

LETTER TO THE EDITOR Will Calcium Supplementation Preserve Bone Integrity? Millions of American women now take calcium supplements in tablet, capsule, pill, or liquid form. Pharmacists report more questions about calcium supplements than about any other over-the-counter medication. It is hoped that increased calcium intakes, up to or beyond the present recommended daily allowance (RDA) will delay, arrest, or even reverse “involutional osteoporosis.” If such calcium supplementation is effective, the prevalence of round back (the dowagers’ hump) should decrease, and the incidence of forearm, femoral neck, trochanteric, and vertebral fractures should go down. Stature shrinkage after the fifth decade might not be an inevitable concomitant of aging, provided that calcium supplementation is effective. Dietary calcium is obviously necessary for bone-building. As much as 100 mg per day may be incorporated into new bone during late-adolescent bone growth. Some workers suggest an even larger amount, though retention of 100 mg per day accounts for 0.15 kg of new bone in a single year. Obviously the amount of dietary calcium necessary to preserve skeletal integrity is more difficult to estimate, since bone loss takes place among older adults in all populations studied, over a wide range of calcium intakes, and in both genders. Decreased estrogen and androgen levels, de26 NUTRITION REVlEWSlVOL 48,NO llJANUARY 1990

creased activity, and changes in absorptive efficiency all complicate attempts to identify calcium intake as the key factor in osteoporosis. New evidence suggests that the age-specific incidence of bone fractures is increasing, at least in Canada, despite dietary improvements in that country.’ The so-called “dietary hypothesis,” has long been associated with the name of Christopher Nordin, and many know it simply as the Nordin hypothesis. Over a period of three decades Nordin has held that an insufficient calcium intake, long maintained, is ultimately responsible for adult bone loss. Years ago Nordin initiated multinational radiographic comparisons, under the auspices of the World Health Organization, to test this hypothesis in places where calcium intakes were low and in regions where they were high. Although Nordin has considered other relevant variables, such as estrogen loss, he continues to reiterate the Nordin hypothesis, effectively summarized in Nufrition Reviews last year.2 At almost the same time, Kanis and P a s ~ m o r e , ~ in a two-part review in the British Medical Journal came to diametrically opposite conclusions. Moreover, a new working paper,4 prepared for the British Nutrition Foundation, similarly rejects the low-calcium hypothesis and the notion that calcium supplementation would be the answer to bone loss in adults. As Kanis and Passmore3 also observed, data on bone-fractures do not support the