- Email: [email protected]
HOMOZYGOUS HYPERCHOLESTERÆMIA AND THE PORTACAVAL SHUNT
449
Occasional
Survey
HOMOZYGOUS HYPERCHOLESTERÆMIA AND THE PORTACAVAL SHUNT The Need for a Concerted Attack
quickly that may guide the course taken by physicians responsible for such patients; and two, to put up for debate a list of high-priority research objectives to which we should give serious consideration preoperatively and postoperatively. Let me discuss each of these two approaches in detail.
by Surgeons and
Clinical Researchers
E. H. AHRENS, JR.
Rockefeller University, New York, N.Y. 10021, U.S.A. Starzl and his colleagues recently reported in these pages1a startling development in the management of a lethal genetic disorder-homozygous familial hypercholestercemia. Five and a half months previously they had performed an end-to-side portacaval shunt in a 12-year-old girl with this disease, and they were now reporting a marked and persistent decrease in plasma-cholesterol levels, resolution of xanthomata, and apparent improvement in coronary blood-flow. Today, 15 months after surgery, her cholesterol levels remain at about 300 mg. per dl. plasma (preoperative levels ranged from 600 to 950); there has been no encephalopathy; and her physical activity seems normal for her age.2 This is a most unusual and unexpected instance of amelioration in a genetic disorder that almost uniformly leads to severe arterial occlusive disease and death before 25 years of age. A wide variety of therapeutic regimens has been applied for control of the rampant hypercholesterxmia that characterises the syndrome: diets rich in polyunsaturated fats and poor in saturates and cholesterol, clofibrate, nicotinic acid, neomycin, cholestyramine, surgical bypass of the distal ileum, and repeated plasmaphereses have usually been disappointing, biochemically and
clinically. The Starzl report has aroused considerable interest among specialists in human lipid metabolism on the practical ground that it seems to offer the possibility of successful management of a uniformly fatal condition. Although the homozygous form of familial hypercholesterasmia is exceedingly rare, it seems likely that, wherever such patients are under observation, this new mode of management will be seriously considered. Since the performance of a portacaval shunt is within the capabilities of surgeons in almost any large medical centre, we can expect to hear within a very few months of patients who have been operated on. But there is great interest also on theoretical grounds: what is the mode of action of portacaval shunt in regulation of cholesterol and/or low-densitylipoprotein metabolism? Any new knowledge gained from studies in operated homozygotes will obviously be of great value in understanding the much more common forms of hypercholesterxmia in heterozygotes and in non-genetic hyperlipidxmias. Thus, in order to take maximum advantage of a truly extraordinary opportunity in clinical research, it seems to me that two approaches are indicated: one, to set up a central registry to which information is sent regarding patients being considered for surgery, and from which replies can be obtained
CE..TRAL
REGISTRY
Dr Robert I. Levy, director of the Division of Heart and Vascular Diseases in the National Heart and
Lung Institute, Bethesda, Maryland, has approached interested investigators in the United States, inviting them to report the contemplation or completion of portacaval shunts to the N.H.L.I. He has made clear that his purpose was to expedite communication, not to endorse the regimen. Formation of such a registry is a step in the right direction. Its value will be greatly enhanced, in my view, if means are found to make this agency rapidly responsive to inquiries from the field. Thus, the experience gained in one centre could be shared quickly with others that are about to undertake this procedure in other patients: clinical advice can be passed along that will benefit the patients, and any new information obtained in earlier-operated cases can be rapidly disseminated, eventually tested, and hopefully amplified in the most informed way by studies mounted in later-operated patients. HIGH-PRIORITY
QUESTIONS
question of highest priority is whether Starzl’s experience can be reproduced in homozygous hypercholestersemic patients who are severely resistant to The
cholesterol-lowering diet/drug regimens. Secondly, the long-term effects of the procedure: does it slow the progression of arteriosclerotic vascular disease (or even promote its regression), without producing harmful long-term effects on liver function or other aspects of metabolic regulation? Then, third, if there is a lowering of plasma-cholesterol levels, we must ask why and how the diversion of portal blood around the liver results in lowered levels of plasmacholesterol. Some of my colleagues consider that it is premature to perform further portacaval shunts in patients with hypercholesteraemia until a better understanding of the long-term effects is obtained through laboratory studies in animals. While I cannot take serious issue with this point of view, I feel certain that shunts will be performed in eligible patients before such laboratory studies are completed. Therefore, I feel it is timely to propose to my medical and surgical colleagues that we act in concert to obtain as many new as we can from the operations that inevitably will be undertaken in the near future. In the following paragraphs I will attempt to describe the kind of studies that may answer the third question posed above: How does this procedure work? I will confine myself to the questions with which I am most familiar, realising that there are many others-in protein and carbohydrate metabolism, particularly.
insights
1. Genetic Validation Epidemiological studies by Patterson and Slack,3 Goldstein et al.,4 Glueck et al.,s Kwiterovich et al.,6 and many
450 others have proven conclusively that genetic factors play an important role in causation of various sorts of hyperlipidsemia. Indeed, the earlier studies of Khachadurian7 had demonstrated that in certain rare individuals with two hypercholestersemic parents the transmission of the genetic trait followed mendelian rules for a dominant monogenic disorder. However, it is argued by Jensen and Blankenhorn,8 Slack,9 Hatch,l° and others that, in the majority of patients with one or another form of hyperlipidaemia, multiple gene effects are manifest. The importance of the distinction is that in the latter case a number of environmental changes can be expected to ameliorate the condition if undertaken together, whereas in the monogenic case the outlook for improvement depends entirely on finding a specific remedy for the specific inherited defect. Starzl’s report1 involved a 12-year-old girl with early onset of hypercholesterxmia of severe degree, xanthomatosis at 6 months of age, onset at 7 years and rapid progression of arteriosclerotic heart-disease, and a family history of premature coronary heart-disease. The diagnosis of homozygous familial hypercholesteraemia is conclusively supported by Goldstein et al.’s studies 11 on cholesterol metabolism in tissue cultures of the skin fibroblasts of Starzl’s patient and her hypercholesteraemic parents (obligate heterozygotes for hypercholesteraemia). Thus, in patients considered for portacaval shunt at this time, it is essential to verify the presence of the truly homozygous state. This validation can be obtained today in only two ways: by detailed studies in first-degree relatives of the index case, preferably in two or more generations, and by tissue-culture studies. Kindred studies must include data on the distribution of hyperlipidsemia, xanthomatosis, and coronary heart-disease, and a search for consanguinity, following the examples set by Patter4 son and Slack 3 and by Goldstein et awl. Special care should be taken to establish that the parents are obligate heterozygotes for hypercholesterxmia per se, and that the index case is not the product of parents who are heterozygotic for two distinctly different lipid disorders,4 for in the latter event it seems likely that the degree of hypercholestersemia is less severe and may be moderately responsive to diet/drug management. Studies of skinfibroblast cultures, as described most recently by Brown and Goldstein,12 should measure the tightness of binding of low-density lipoprotein (L.D.L.) to normal and suspect cells, as well as the degree of degradation of L.D.L. on exposure to such cells, and lack of feedback control of a key regulatory enzyme in cholesterol biosynthesis, H.M.G.-CoA reductase (f3-hydroxy-/3-methylglutarylcoenzyme A reductase). 2. Cholesterol Homceostasis A key observation in Starzl’s reportwas the slow but steady decline in plasma-cholesterol levels postoperatively, from preoperative levels of about 700 mg. per dl. plasma to a new plateau of about 250 mg. by the sixth month. These low levels have been maintained for the last 8 months. Was this due to a decrease in daily cholesterol synthesis; to decreased absorption of cholesterol ; or to increased conversion of cholesterol to bile acids? Since skin and tendon xanthomata were reported to be resolving, it seems unlikely that the content of cholesterol in the plasma was being reduced by its transfer into tissue storage sites, yet this possibility cannot be discarded out of hand. Sterol balance methods perfected over the past decade 13 allow us to measure the rates of daily cholesterol synthesis and conversion of cholesterol to bile acids. The metabolic steady state is required-i.e., the input and efflux of cholesterol from the body must be equal. Methods that permit the measurement of the hourly flow-rates of all
the biliary lipids (cholesterol, bile acids, and lecithin) out of the liver also have been devised.14 Thus, it now is feasible, without recourse to use of radioactive materials, to estimate whether the patient subjected to portacaval shunt shows an increased excretion of cholesterol and bile acids postoperatively, and to determine whether there is an increased or decreased flux of cholesterol and bile acids through the biliary tree or urinary tract. However, unless radioactive materials are employed, it will not be possible in the postoperative state to distinguish, by balance methods alone, increased synthesis of cholesterol from its increased efflux from tissues. The increased efflux that might be expected to occur for many months postoperatively is a condition of the unsteady state, and in that condition the results obtained by balance methods have limited usefulness. We now have reason to believe that measurements of sterol-synthesis rates in such unsteady metabolic states are obtainable through the use of radioisotopic methods developed in this laboratory in the past year.l5 Other applications of isotopic techniques also can provide further essential data: (a) estimates of pool sizes of cholesterol, before and after operation 16; (b) measurements of daily rates of cholesterol absorption 17; (c) measurements of daily rates of synthesis and of pool sizes of the primary bile acids (cholic and chenodeoxycholic acids) 18; (d) estimates of the disposition of absorbed dietary cholesterol (through excretion of neutral steroids, conversion to bile acids, reduction of cholesterol synthesis, or retention in tissue stores).19 3. Kinetics
of Low-density-lipoprotein Metabolism Homozygous hypercholesteraemia is characterised by elevated levels of circulating L.D.L. Goldstein and Brown 20 suggest that in patients with this disorder these high levels are required in order to feedback-control the rate of cholesterol synthesis at the not-too-abnormal rates we have found 21 in the metabolic steady state in these patients. it has become feasible 22.23 to measure the and pool-sizes of L.D.L. labelled with 1251. Where such studies can be carried out, it will be of great interest to determine the direction in which L.D.L. synthesis is altered by routing portal blood around the liver, and whether the procedure causes an increase in catabolism of L.D.L. by peripheral tissues supplied with blood richer than normal in pancreatic hormones or in bile acids. It is of interest that Sniderman et a1.23 have reported that hepatectomy in dogs leads to an increased catabolism of labelled L.D.L. by peripheral tissues. In
recent years
turnover-rates
4. Liver Function and
Morphology
Liver-function tests, before and repeatedly after operation, are obviously indicated, and will not be considered here further. Starzlreported that a punch biopsy of liver 6 months postoperatively showed a significant reduction in the amount of rough endoplasmic reticulum; he suggested that cholesterol synthesis was reduced as a result. Earlier experiments in dogs dating back to those of Rous and Larimore 24 in 1920 and subsequently greatly extended by Starzl et a125 in 1973have shown a marked inhibitory effect on liver regeneration by supplying it with nutrients coming from the intestine, as compared to blood arriving from the pancreatic bed. In view of this background it would seem useful to scan the size of the liver before operation and periodically after operation to determine whether reductions in size result from portacaval shunting. The histology of the liver should be judged by routine methods and by electron microscopy at the time of surgery, and subsequently, if possible, by needle aspiration biopsy, with an attempt at quantification of subcellular organelles, as described by
451
all and of lipoprotein particles in the Golgi described by Jones et all Enzyme studies liver tissue in patients with hyperlipidaemia are proving
Weibel
et
apparatus on
Public Health
as
be highly informative: it would be desirable to assay the activities of key enzymes in cholesterol and bile-acid synthesis (H.M.G.-CoA reductase and cholesterol 7ahydroxylase), as recently described by Shefer et al.28 to
5.
Carbohydrate Metabolism
HIGH FREQUENCY OF ANTIBIOTICRESISTANT ENTEROBACTERIA IN THE RIVER STOUR, KENT COLIN HUGHES
Starzl’s animal studies 25 led him to conclude that the
key change created by the portacaval shunt is the routing of insulin and glucagon away from liver to the peripheral tissues. In patients with cirrhosis it has been reported by Sherwin et a1.29 that glucagon and insulin levels in peripheral blood are elevated after completion of a portacaval shunt. It will be of interest to measure the usual parameters of carbohydrate metabolism in homozygous hypercholesteraemics before and after surgery: oral glucose tolerance, intravenous glucose kinetics, and coincident levels of insulin and glucagon. But it would be even more revealing to be able to measure the pool-sizes and turnover-rates of these hormones, in order to determine whether hormone production and catabolism are disturbed by the redistribution of pancreatic blood. CONCLUSION
Much can be gained by pooling ideas and plans for intervention, and the data obtained at surgery. And so I hope that those who recognise the potentialities of Starzl’s discovery will work together to make the greatest capital from it. We owe this to the homozygous hypercholesteraemic patients whose lives are threatened at a shockingly early age, and to the many others in whom the acquisition of a new understanding may direct our future efforts in prevention of premature coronary heart-disease. This laboratory is supported in part by U.S. Public Health Service grant HL-06222 from the National Heart and Lung Institute, and by U.S. Public Health Service grant FR-00102 from the general clinical research centers branch of the Division of Research Resources. REFERENCES
Starzl, T. E., Chase, H. P., Putnam, C. 1973, ii, 940. 2. Starzl, T. E. Personal communication. 1.
W., Porter, K. A. Lancet,
Patterson, D., Slack, J. Lancet, 1972, i, 393. Goldstein, J. L., Hazzard, W. R., Schrott, H. G., Bierman, E. L., Motulsky, A. G. J. clin. Invest. 1973, 52, 1533. 5. Glueck, C. J., Fallat, R. W., Tsang, R., Buncher, C. R. Am. J. Dis. Child. 1974, 127, 70. 6. Kwiterovich, P. O., Jr., Fredrickson, D. S., Levy, R. I. J. clin. Invest. 1974, 53, 1237. 7. Khachadurian, A. Am. J. Med. 1964, 37, 402. 8. Jensen, J., Blankenhorn, D. H. ibid. 1972, 52, 499. 9. Slack, J. J. R. Coll. Physns Lond. 1974, 8, 115. 10. Hatch, F. T. Am. J. clin. Nutr. 1974, 27, 80. 11. Goldstein, J. L., Harrod, M. J. E., Brown, M. S. Am. J. hum. Genet. 1974, 26, 199. 12. Brown, M. S., Goldstein, J. L. Proc. natn. Acad. Sci. U.S.A. 1974, 71, 788. 13. Grundy, S. M., Ahrens, E. H., Jr. J. Lipid Res. 1969, 10, 91. 14. Grundy, S. M., Metzger, A. L. Gastroenterology, 1972, 62, 1200. 15. Liu, G. C. K., Schreibman, P. H., Samuel, P., Ahrens, E. H., Jr. J. clin. Invest. 1974, 53, 47a. 16. Samuel, P., Lieberman, S. J. Lipid Res. 1973, 14, 189. 17. Quintão, E., Grundy, S. M., Ahrens, E. H., Jr. ibid. 1971, 12, 221. 18. Vlahcevic, Z. R., Bell, C. C., Jr., Gregory, D. H., Buker, G., Juttijudata, P., Swell, L. Gastroenterology, 1972, 62, 73. 19. Quintão, E., Grundy, S. M., Ahrens, E. H., Jr. J. Lipid Res. 1971, 12, 233. 20. Goldstein, J. L., Brown, M. S. Clin. Res. 1974, 22, 559A. 21. Grundy, S. M., Ahrens, E. H., Jr., Salen, G. J. Lab. clin. Med. 1971, 78, 94. 22. Langer, T., Strober, W., Levy, R. I. J. clin. Invest. 1972, 51, 1528. 3. 4.
G. G. MEYNELL
Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ The proportion of antibiotic-resistant enterobacteria in natural waters seems to be increasing. For example, in one river in 1974 73% of the enterobacteria isolated were ampicillin resistant, whereas in another study in 1970 27% of organisms isolated from other rivers were ampicillin resistant. The number of antibiotic-resistant enterobacteria is underestimated in colony-counts using pour plates.
Summary
INTRODUCTION
IT is to be expected that, as antibiotic resistance increases in the enterobacteria of man and animals, resistant organisms will increasingly be found in natural waters. By 1970-71 a substantial proportion of organisms from various rivers and coastal waters of the U.K. were already resistant, and many strains could transfer their resistance in mixed cultures We have determined the frequency of antibioticresistant enterobacteria in the River Stour, Kent, this year. METHODS
Samples.-Six sites
were examined. Samples I and II I was taken at the collected on Jan. 23, 1974: northern edge of Canterbury (national grid reference, TR 134578) and II (TR 175599) about 3 miles downstream and about 1 mile downstream from a sewage works. Samples iii-vi were collected on Feb. 8, 1974: III came 2-5 miles upstream from Sandwich (TR 322596); iv and v came 2 and 2’5 miles, respectively, downstream from Sandwich (TR 335612, TR 337618); and vi came from the shoreline of Pegwell Bay where the Stour enters the were
(TR 350641). Colony-counts.-These were made within 3 hours of collecting the samples, using Oxoid MacConkey No. 3 as supplied or with streptomycin, tetracycline, ampicillin, or chloramphenicol at 20 gg. per ml. agar each. Counts were made either by a drop method 3 or by pour plates in which a 1 ml. sample was added to 20 ml. molten agar at 48 °C which was poured immediately and allowed to sea
Colonies room-temperature. incubation 37 °C. at overnight set at
were
counted after
23. Sniderman, A. L., Carew, T. E., Chandler, J. G., Steinberg, D. Science, 1974, 183, 526. 24. Rous, P., Larimore, L. D. J. exp. Med. 1920, 31, 609. 25. Starzl, T. E., Francavilla, A., Halgrimson, C. G., Francavilla, F. R., Porter, K. A., Brown, T. H., Putnam, C. W. Surgery Gynec. Obstet. 1973, 137, 179. 26. Weibel, E. R., Staubli, W., Gnägi, H. R., Hess, F. A. J. Cell Biol. 27. 28. 29.
1969, 42, 69. Jones, A. L., Ruderman, N. B., Herrera, M. G. J. Lipid Res. 1967, 8, 429. Shefer, S., Hauser, S., Lapar, V., Mosbach, E. H. ibid. 1973, 14, 573. Sherwin, R., Joshi, P., Hendler, R., Felig, P., Conn, H. O. New Engl. J. Med. 1974, 290, 239.
Occasional
Survey
HOMOZYGOUS HYPERCHOLESTERÆMIA AND THE PORTACAVAL SHUNT The Need for a Concerted Attack
quickly that may guide the course taken by physicians responsible for such patients; and two, to put up for debate a list of high-priority research objectives to which we should give serious consideration preoperatively and postoperatively. Let me discuss each of these two approaches in detail.
by Surgeons and
Clinical Researchers
E. H. AHRENS, JR.
Rockefeller University, New York, N.Y. 10021, U.S.A. Starzl and his colleagues recently reported in these pages1a startling development in the management of a lethal genetic disorder-homozygous familial hypercholestercemia. Five and a half months previously they had performed an end-to-side portacaval shunt in a 12-year-old girl with this disease, and they were now reporting a marked and persistent decrease in plasma-cholesterol levels, resolution of xanthomata, and apparent improvement in coronary blood-flow. Today, 15 months after surgery, her cholesterol levels remain at about 300 mg. per dl. plasma (preoperative levels ranged from 600 to 950); there has been no encephalopathy; and her physical activity seems normal for her age.2 This is a most unusual and unexpected instance of amelioration in a genetic disorder that almost uniformly leads to severe arterial occlusive disease and death before 25 years of age. A wide variety of therapeutic regimens has been applied for control of the rampant hypercholesterxmia that characterises the syndrome: diets rich in polyunsaturated fats and poor in saturates and cholesterol, clofibrate, nicotinic acid, neomycin, cholestyramine, surgical bypass of the distal ileum, and repeated plasmaphereses have usually been disappointing, biochemically and
clinically. The Starzl report has aroused considerable interest among specialists in human lipid metabolism on the practical ground that it seems to offer the possibility of successful management of a uniformly fatal condition. Although the homozygous form of familial hypercholesterasmia is exceedingly rare, it seems likely that, wherever such patients are under observation, this new mode of management will be seriously considered. Since the performance of a portacaval shunt is within the capabilities of surgeons in almost any large medical centre, we can expect to hear within a very few months of patients who have been operated on. But there is great interest also on theoretical grounds: what is the mode of action of portacaval shunt in regulation of cholesterol and/or low-densitylipoprotein metabolism? Any new knowledge gained from studies in operated homozygotes will obviously be of great value in understanding the much more common forms of hypercholesterxmia in heterozygotes and in non-genetic hyperlipidxmias. Thus, in order to take maximum advantage of a truly extraordinary opportunity in clinical research, it seems to me that two approaches are indicated: one, to set up a central registry to which information is sent regarding patients being considered for surgery, and from which replies can be obtained
CE..TRAL
REGISTRY
Dr Robert I. Levy, director of the Division of Heart and Vascular Diseases in the National Heart and
Lung Institute, Bethesda, Maryland, has approached interested investigators in the United States, inviting them to report the contemplation or completion of portacaval shunts to the N.H.L.I. He has made clear that his purpose was to expedite communication, not to endorse the regimen. Formation of such a registry is a step in the right direction. Its value will be greatly enhanced, in my view, if means are found to make this agency rapidly responsive to inquiries from the field. Thus, the experience gained in one centre could be shared quickly with others that are about to undertake this procedure in other patients: clinical advice can be passed along that will benefit the patients, and any new information obtained in earlier-operated cases can be rapidly disseminated, eventually tested, and hopefully amplified in the most informed way by studies mounted in later-operated patients. HIGH-PRIORITY
QUESTIONS
question of highest priority is whether Starzl’s experience can be reproduced in homozygous hypercholestersemic patients who are severely resistant to The
cholesterol-lowering diet/drug regimens. Secondly, the long-term effects of the procedure: does it slow the progression of arteriosclerotic vascular disease (or even promote its regression), without producing harmful long-term effects on liver function or other aspects of metabolic regulation? Then, third, if there is a lowering of plasma-cholesterol levels, we must ask why and how the diversion of portal blood around the liver results in lowered levels of plasmacholesterol. Some of my colleagues consider that it is premature to perform further portacaval shunts in patients with hypercholesteraemia until a better understanding of the long-term effects is obtained through laboratory studies in animals. While I cannot take serious issue with this point of view, I feel certain that shunts will be performed in eligible patients before such laboratory studies are completed. Therefore, I feel it is timely to propose to my medical and surgical colleagues that we act in concert to obtain as many new as we can from the operations that inevitably will be undertaken in the near future. In the following paragraphs I will attempt to describe the kind of studies that may answer the third question posed above: How does this procedure work? I will confine myself to the questions with which I am most familiar, realising that there are many others-in protein and carbohydrate metabolism, particularly.
insights
1. Genetic Validation Epidemiological studies by Patterson and Slack,3 Goldstein et al.,4 Glueck et al.,s Kwiterovich et al.,6 and many
450 others have proven conclusively that genetic factors play an important role in causation of various sorts of hyperlipidsemia. Indeed, the earlier studies of Khachadurian7 had demonstrated that in certain rare individuals with two hypercholestersemic parents the transmission of the genetic trait followed mendelian rules for a dominant monogenic disorder. However, it is argued by Jensen and Blankenhorn,8 Slack,9 Hatch,l° and others that, in the majority of patients with one or another form of hyperlipidaemia, multiple gene effects are manifest. The importance of the distinction is that in the latter case a number of environmental changes can be expected to ameliorate the condition if undertaken together, whereas in the monogenic case the outlook for improvement depends entirely on finding a specific remedy for the specific inherited defect. Starzl’s report1 involved a 12-year-old girl with early onset of hypercholesterxmia of severe degree, xanthomatosis at 6 months of age, onset at 7 years and rapid progression of arteriosclerotic heart-disease, and a family history of premature coronary heart-disease. The diagnosis of homozygous familial hypercholesteraemia is conclusively supported by Goldstein et al.’s studies 11 on cholesterol metabolism in tissue cultures of the skin fibroblasts of Starzl’s patient and her hypercholesteraemic parents (obligate heterozygotes for hypercholesteraemia). Thus, in patients considered for portacaval shunt at this time, it is essential to verify the presence of the truly homozygous state. This validation can be obtained today in only two ways: by detailed studies in first-degree relatives of the index case, preferably in two or more generations, and by tissue-culture studies. Kindred studies must include data on the distribution of hyperlipidsemia, xanthomatosis, and coronary heart-disease, and a search for consanguinity, following the examples set by Patter4 son and Slack 3 and by Goldstein et awl. Special care should be taken to establish that the parents are obligate heterozygotes for hypercholesterxmia per se, and that the index case is not the product of parents who are heterozygotic for two distinctly different lipid disorders,4 for in the latter event it seems likely that the degree of hypercholestersemia is less severe and may be moderately responsive to diet/drug management. Studies of skinfibroblast cultures, as described most recently by Brown and Goldstein,12 should measure the tightness of binding of low-density lipoprotein (L.D.L.) to normal and suspect cells, as well as the degree of degradation of L.D.L. on exposure to such cells, and lack of feedback control of a key regulatory enzyme in cholesterol biosynthesis, H.M.G.-CoA reductase (f3-hydroxy-/3-methylglutarylcoenzyme A reductase). 2. Cholesterol Homceostasis A key observation in Starzl’s reportwas the slow but steady decline in plasma-cholesterol levels postoperatively, from preoperative levels of about 700 mg. per dl. plasma to a new plateau of about 250 mg. by the sixth month. These low levels have been maintained for the last 8 months. Was this due to a decrease in daily cholesterol synthesis; to decreased absorption of cholesterol ; or to increased conversion of cholesterol to bile acids? Since skin and tendon xanthomata were reported to be resolving, it seems unlikely that the content of cholesterol in the plasma was being reduced by its transfer into tissue storage sites, yet this possibility cannot be discarded out of hand. Sterol balance methods perfected over the past decade 13 allow us to measure the rates of daily cholesterol synthesis and conversion of cholesterol to bile acids. The metabolic steady state is required-i.e., the input and efflux of cholesterol from the body must be equal. Methods that permit the measurement of the hourly flow-rates of all
the biliary lipids (cholesterol, bile acids, and lecithin) out of the liver also have been devised.14 Thus, it now is feasible, without recourse to use of radioactive materials, to estimate whether the patient subjected to portacaval shunt shows an increased excretion of cholesterol and bile acids postoperatively, and to determine whether there is an increased or decreased flux of cholesterol and bile acids through the biliary tree or urinary tract. However, unless radioactive materials are employed, it will not be possible in the postoperative state to distinguish, by balance methods alone, increased synthesis of cholesterol from its increased efflux from tissues. The increased efflux that might be expected to occur for many months postoperatively is a condition of the unsteady state, and in that condition the results obtained by balance methods have limited usefulness. We now have reason to believe that measurements of sterol-synthesis rates in such unsteady metabolic states are obtainable through the use of radioisotopic methods developed in this laboratory in the past year.l5 Other applications of isotopic techniques also can provide further essential data: (a) estimates of pool sizes of cholesterol, before and after operation 16; (b) measurements of daily rates of cholesterol absorption 17; (c) measurements of daily rates of synthesis and of pool sizes of the primary bile acids (cholic and chenodeoxycholic acids) 18; (d) estimates of the disposition of absorbed dietary cholesterol (through excretion of neutral steroids, conversion to bile acids, reduction of cholesterol synthesis, or retention in tissue stores).19 3. Kinetics
of Low-density-lipoprotein Metabolism Homozygous hypercholesteraemia is characterised by elevated levels of circulating L.D.L. Goldstein and Brown 20 suggest that in patients with this disorder these high levels are required in order to feedback-control the rate of cholesterol synthesis at the not-too-abnormal rates we have found 21 in the metabolic steady state in these patients. it has become feasible 22.23 to measure the and pool-sizes of L.D.L. labelled with 1251. Where such studies can be carried out, it will be of great interest to determine the direction in which L.D.L. synthesis is altered by routing portal blood around the liver, and whether the procedure causes an increase in catabolism of L.D.L. by peripheral tissues supplied with blood richer than normal in pancreatic hormones or in bile acids. It is of interest that Sniderman et a1.23 have reported that hepatectomy in dogs leads to an increased catabolism of labelled L.D.L. by peripheral tissues. In
recent years
turnover-rates
4. Liver Function and
Morphology
Liver-function tests, before and repeatedly after operation, are obviously indicated, and will not be considered here further. Starzlreported that a punch biopsy of liver 6 months postoperatively showed a significant reduction in the amount of rough endoplasmic reticulum; he suggested that cholesterol synthesis was reduced as a result. Earlier experiments in dogs dating back to those of Rous and Larimore 24 in 1920 and subsequently greatly extended by Starzl et a125 in 1973have shown a marked inhibitory effect on liver regeneration by supplying it with nutrients coming from the intestine, as compared to blood arriving from the pancreatic bed. In view of this background it would seem useful to scan the size of the liver before operation and periodically after operation to determine whether reductions in size result from portacaval shunting. The histology of the liver should be judged by routine methods and by electron microscopy at the time of surgery, and subsequently, if possible, by needle aspiration biopsy, with an attempt at quantification of subcellular organelles, as described by
451
all and of lipoprotein particles in the Golgi described by Jones et all Enzyme studies liver tissue in patients with hyperlipidaemia are proving
Weibel
et
apparatus on
Public Health
as
be highly informative: it would be desirable to assay the activities of key enzymes in cholesterol and bile-acid synthesis (H.M.G.-CoA reductase and cholesterol 7ahydroxylase), as recently described by Shefer et al.28 to
5.
Carbohydrate Metabolism
HIGH FREQUENCY OF ANTIBIOTICRESISTANT ENTEROBACTERIA IN THE RIVER STOUR, KENT COLIN HUGHES
Starzl’s animal studies 25 led him to conclude that the
key change created by the portacaval shunt is the routing of insulin and glucagon away from liver to the peripheral tissues. In patients with cirrhosis it has been reported by Sherwin et a1.29 that glucagon and insulin levels in peripheral blood are elevated after completion of a portacaval shunt. It will be of interest to measure the usual parameters of carbohydrate metabolism in homozygous hypercholesteraemics before and after surgery: oral glucose tolerance, intravenous glucose kinetics, and coincident levels of insulin and glucagon. But it would be even more revealing to be able to measure the pool-sizes and turnover-rates of these hormones, in order to determine whether hormone production and catabolism are disturbed by the redistribution of pancreatic blood. CONCLUSION
Much can be gained by pooling ideas and plans for intervention, and the data obtained at surgery. And so I hope that those who recognise the potentialities of Starzl’s discovery will work together to make the greatest capital from it. We owe this to the homozygous hypercholesteraemic patients whose lives are threatened at a shockingly early age, and to the many others in whom the acquisition of a new understanding may direct our future efforts in prevention of premature coronary heart-disease. This laboratory is supported in part by U.S. Public Health Service grant HL-06222 from the National Heart and Lung Institute, and by U.S. Public Health Service grant FR-00102 from the general clinical research centers branch of the Division of Research Resources. REFERENCES
Starzl, T. E., Chase, H. P., Putnam, C. 1973, ii, 940. 2. Starzl, T. E. Personal communication. 1.
W., Porter, K. A. Lancet,
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G. G. MEYNELL
Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ The proportion of antibiotic-resistant enterobacteria in natural waters seems to be increasing. For example, in one river in 1974 73% of the enterobacteria isolated were ampicillin resistant, whereas in another study in 1970 27% of organisms isolated from other rivers were ampicillin resistant. The number of antibiotic-resistant enterobacteria is underestimated in colony-counts using pour plates.
Summary
INTRODUCTION
IT is to be expected that, as antibiotic resistance increases in the enterobacteria of man and animals, resistant organisms will increasingly be found in natural waters. By 1970-71 a substantial proportion of organisms from various rivers and coastal waters of the U.K. were already resistant, and many strains could transfer their resistance in mixed cultures We have determined the frequency of antibioticresistant enterobacteria in the River Stour, Kent, this year. METHODS
Samples.-Six sites
were examined. Samples I and II I was taken at the collected on Jan. 23, 1974: northern edge of Canterbury (national grid reference, TR 134578) and II (TR 175599) about 3 miles downstream and about 1 mile downstream from a sewage works. Samples iii-vi were collected on Feb. 8, 1974: III came 2-5 miles upstream from Sandwich (TR 322596); iv and v came 2 and 2’5 miles, respectively, downstream from Sandwich (TR 335612, TR 337618); and vi came from the shoreline of Pegwell Bay where the Stour enters the were
(TR 350641). Colony-counts.-These were made within 3 hours of collecting the samples, using Oxoid MacConkey No. 3 as supplied or with streptomycin, tetracycline, ampicillin, or chloramphenicol at 20 gg. per ml. agar each. Counts were made either by a drop method 3 or by pour plates in which a 1 ml. sample was added to 20 ml. molten agar at 48 °C which was poured immediately and allowed to sea
Colonies room-temperature. incubation 37 °C. at overnight set at
were
counted after
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1969, 42, 69. Jones, A. L., Ruderman, N. B., Herrera, M. G. J. Lipid Res. 1967, 8, 429. Shefer, S., Hauser, S., Lapar, V., Mosbach, E. H. ibid. 1973, 14, 573. Sherwin, R., Joshi, P., Hendler, R., Felig, P., Conn, H. O. New Engl. J. Med. 1974, 290, 239.