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METHOD FOR ESTIMATING RATE OF FAT LOSS DURING TREATMENT OF OBESITY BY CALORIE RESTRICTION
482 METHOD FOR ESTIMATING RATE OF FAT LOSS DURING TREATMENT OF OBESITY BY CALORIE RESTRICTION V. WYNN
R. R. ABRAHAM
metabolic balance measurements, has enabled us to interpret the daily weight-loss pattern of patients, to become rapidly alerted to non-compliance, and to compare different dietary regimens over long periods. We have studied twenty-five obese patients during calorie restriction for up to 120 days.
J. W. DENSEM Alexander Simpson Laboratory for Metabolic Research, St Mary’s Hospital Medical School, London W2
Rates of weight and fat loss in sixteen female and nine male obese patients during calorie restriction (655-789 kcal/day) for up to 120 days were studied by a method for estimating daily changes in body composition. Fat mass is calculated by subtracting daily fluid (calculated from sodium and potassium balances) and protein mass changes from daily weight changes. After the natriuresis of the first 4 days, there was a slower rate of weight loss between days 5 and 28 due largely to a decreasing contribution from fluid and protein losses. No significant change in the rate of weight loss could be shown after the first 28 days. The rate of fat loss did not change significantly from day 5 onwards suggesting no significant change in total energy expenditure during the study period. After 68 days, 93·4% of the weight loss was fat.
Summary
Introduction THE difficulty in losing. weight reported by many obese patients after a few weeks on calorie-restricted diets may be the result of various adaptive mechanisms, such as reduced fluid and protein losses and metabolic rates as well as dietary non-compliance and fluid retention. To assess the role of these responses the composition of the weight lost during dieting must be known. Changes in fluid balance and body protein must be accounted for before ascribing weight loss to
fat. We have developed a method for the measurement of the main components of weight change during dieting. Labile carbohydrate stores (glycogen) are small and are depleted within a few days when energy intake is reduced,I,5 making no further contribution to weight loss. Changes in body water are estimated from the sum of external sodium and potassium balances and protein changes from nitrogen balance. From these data, it is possible to estimate the daily fat loss. Other indirect methods of measuring body fat are invasive, complicated, or not suitable for continuous monitoring of the patient. Our method, using strict dietary controls and
7. Hirata
Y, Ishizu H. Elevated insulin-binding capacity of serum proteins in a case of spontaneous hypoglycemia and mild diabetes not treated with insulin. Tokohu J Exp Med 1972; 107: 277-86. 8. Foiling I, Norman N. Hyperglycemia, hypoglycemic attacks and production of antiinsulin antibodies without previous known immunisation. Diabetes 1972, 21: 814-26. 9. Goldman J, Baldwin D, Rubenstein AH et al. Characterisation of circulating insulin and pro-insulin binding antibodies in autoimmune hypoglycemia. J Clin Invest 1979; 63: 1050-59. 10. Palmer JP, Asplin CM, Clemons P, et al. Insulin antibodies in insulin-dependent diabetics before insulin treatment. Science 1983; 222: 1337-39. 11. Wilkin TJ, Nicholson S. Autoantibodies against human insulin. Br Med J 1984; 288: 349-52. 12. Reeves WG. Insulin antibody determination: Theoretical and practical considerations. Diabetologia 1983; 24: 399-403. 13. Spencer KM, Tarn A, Dean BM, Lister J, Bottazzo GF. Fluctuating islet-cell autoimmunity in unaffected relatives of patients with insulin-dependent diabetes. Lancet 1984; i. 764-66. 14. Wilkin TJ, Swanson Beck J, Gunn A, Newton RW, Isles TE, Crookes J. Autoantibodies in thyrotoxicosis. A quantitative study of their behaviour in relation to the course and outcome of treatment. J Endocrinol Invest 1980; 3: 5-14. 15. Wright R Immunology of gastrointestinal and liver disease London: Edward Arnold, 1977: 16-26.
Patients and Methods Patients The mean age of the nine male patients was 38 - 6-t4 -years and their mean weight was 137-7±9-33 kg (196±16% of ideal6). The mean age of the sixteen women was 38 - 2±4 - 0 years and their mean weight was 98 - 9±4 - 8 kg (178±9% ofidear). All were clinically and biochemically euthyroid. Patients who had lost weight before admission or were on diuretic or thyroid therapy were excluded. After fasting overnight and emptying the bladder, each patient, wearing a gown, was weighed on an electronic balance with a precision of <10 g in 100 kg (coefficient of variation 0-0121%). Patients weighing more than 140 kg were weighed on a beam balance with manual zeroing and a precision of 28 g in 120 kg (coefficient of variation 0-024%). Patients did not take strenuous physical exercise but their activities on the ward were not restricted.
Electrolyte and Nitrogen Balance Patients were studied in the metabolic ward with rigorous attention to dietary intake and 24 h urine and stool collections. Lowcalorie liquid formula and mixed diets ranging between 655 and 789 kcal energy content (50-53 g protein, 37-58 mmol sodium, 49-66 mmol potassium, 30-87 g carbohydrate, 11-37 g fat, and 2-31 g fibre) were prepared from bulk supplies by a consistent technique; the composition will be reported elsewhere. Diets for weight maintenance or weight gain were prepared using ’Nutrauxil’ (Kabivitrum, Uxbridge), ’Prosparol’ (50:50 arachis oil in water, Duncan Flockhart, London), and ’Polycal’ (maltodextrins, Nutricia, The Netherlands). Vitamin and iron supplements were provided, with appropriate sodium and potassium chloride additions. The diets were analysed in triplicate monthly. Drinking water was deionised. Dietary energy content was measured by adiabatic bomb calorimetry. Flame photometry and the Kjeldahl method were used to measure the daily excretion of electrolytes and nitrogen in urine and stool samples. The latter were stored in weekly batches for analysis. The between-assay coefficients of variation were less than 3-9/0. An allowance for blood samples taken and heparinised saline infused during diagnostic tests was made for each patient. We used currently accepted figures for integumental and sweat losses (3 mmol/day sodium,2mmol/day potassium,8 and O.3 g/day nitrogen9>o), confirmed as reasonable estimates by our own measurements of sweat loss on a separate group of eight patients by the method of Ashley and Whyte." The total mean daily unmeasured losses (integumental and venesection) ranged between 5 - 5 and 9 - 3 mmol for sodium plus potassium and were 0 - 78±0’ 03 g/day for nitrogen. Mean faecal energy losses, measured by bomb calorimetry, 12 were 58 - 3±7 - 8 kcal/day nearly 9% (range, 7-12%) of the energy intake.
Assessment
of Components of Weight Loss
Body water. -Electrolytes constitute about 98% of the osmotically active solutes in body fluids.13,14 Since extracellular and intracellular fluids are in osmotic equilibrium, changes in electrolyte balance can be taken to reflect changes in body water provided extracellular osmolality remains constant.13 Total body water is closely correlated with body sodium and potassium (measured by isotope dilution) both in health and disease. We used the data of Edelman et al 15 to obtain the sum of sodium plus potassium associated with a litre of body water (153-6±5-66 mmol). Direct measurements of the change in total body water were obtained in fourteen patients by tritiated water dilution with rapid vacuum sublimation’-6 of urine, blood, and saliva.
483 TABLE 1-MEAN+SEM METABOLIC BALANCE DATA
For differences between 5-28 days and 28-68 days: *p<0.001; p<0.005. $In the ten patients studied for longer than 68 days, these percentages for day 68 to end of diet were: fat 93 - 4%, fluid I - 6%, protein 5 - 0%.
Fig 1-Mean rates of weight and fat loss. 4-day means are shown.
Body protein changes were calculated from using a factor of 6-25.
the
net
nitrogen
balance
Body fat changes were calculated by subtracting from the weight changes the body water and protein changes. Predicted Change in Absorbed Energy
Daily Fat Mass from Known Change in
Assuming constant daily energy expenditure on two diets of different energy content, the predicted change in daily fat mass was calculated by dividing the difference in the absorbed energy of the two diets by 9-3 3 kcal/g, the heat of combustion of animal fat. 17 Energy lost as protein was subtracted (using 5 - 65 kcal/g as the heat of combustion of muscle protein 17). Energy equivalents of 10-66 kcal/g and 12 kcal/g for fat and protein, respectively, were used when there was net storage. 18,199 Resting energy expenditure (REE) was measured by indirect calorimetry with a face mask. Argon dilution was used to measure minute volume and gas concentrations were measured with an MSR2 Medishield mass spectrometer. 20 The REE was calculated from the modification introduced by Weir 21 using the non-protein
day 28, the cumulative mean weight loss was linear (r = 0 - 99969, p<0. 001) (table I). For days 5 to 68, the cumulative mean rate of fat loss was linear (r = 0 99897, p<0.001; fig 1); the rate was higher in men than in women (p<0 . table 001; t). For twelve of thirteen 120 studied for to subjects up days, there was no further in rate of weight or fat loss. significant change An example of the use of our method to calculate the rate of fat loss is shown for a male patient in fig 2. The rate of fat, loss (298±18 g/day) was 90% of the rate of weight loss (330±19 g/day) and did not change significantly with time. When weight loss is not complicated by large changes in fluid balance, the rate of loss of fat becomes more closely related to the rate of weight loss as protein loss approaches an equilibrium during long periods of calorie restriction.
respiratory quotient. Statistics All results are expressed as the mean with the standard Student’s t test was used for comparisons.
error.
Results All the patients were on continuous calorie restriction for at least 28 days. Six men and seven women continued dieting for at least 68 days. In both men and women, there was an initial rapid weight loss (men 735± 102 g/day, women 477±71 g/day for days 1-4) which was significantly greater than that during days 5-8 (men 466±57 g/day, p<0.05; women 273±23 g/day, p<0.01) (fig 1). Between days 5 and 28 there was a significant exponential decrease in the daily weight loss in both groups (by 1 - 60% of the daily rate of weight loss, p<0-05); by days 25-28, the rate of weight loss was 334±33 g/day in men and 195±30 g/day in women (p<0 . 05). After
Fig 2-Cumulative weight and calculated fat loss and sodium, potassium, and nitrogen balances of patient 1 (male, aged 49 years, height 180 cm, 235% ideal body weight.)
484
Fig 4-Metabolic balance study of patient 2 (female, aged height 139-0 cm, 187% ideal body weight).
Fig 3-Daily fluid, sodium, potassium,
and
nitrogen balance.
Fluid loss in men and women was most closely associated with sodium loss in the first week, especially on the first day (fig 3). Between days 5 and 28 there was a significant fall in the rate of fluid loss by 6 - 77 g/day (p<0 . 00 I) in men and 1.93 in the mean fluid loss over this time g/day (p<0. 0 1) women, in men in than women (p<0. 01, being significantly greater table I). The fluid loss during days 5-28 is mainly a result of the continuing intracellular potassium losses that accompany protein loss and its amount and duration are greater in men. Nitrogen loss was consistently greater in men than in women (p<0’001, table I); neither group achieved zero nitrogen balance which decreased by 0-19 g/day in men and 0’ 10 g/day in women between days 5-28. Potassium loss in men and women was related to the continuing nitrogen deficit (2’337mmol potassium/g nitrogen). Until the rate of weight loss stabilised after about 28 days of calorie restriction an increasing percentage of fat and a
50 years,
of water and protein were lost (table r). The percentage of fat in the weight lost after stabilisation estimated by our method (87-93%) agreed closely with the 92 - 5% obtained from total body water measurements (assuming 73-2% water in the fat-free mass 22) in seven patients studied between 61 and 114 days of continuous calorie restriction with our diets; mean weight loss was
reducing percentage
15-02
kg.
Influence of Diuretics and Fluid Balance Changes on Calculated Rate of Fat Loss The ability to distinguish between weight loss due to changes in fluid balance and that due to-fat loss is crucial to the use of our method. The failure of large changes of fluid balance to influence substantially the cumulative fat loss slope is illustrated by an individiaul metabolic balance study (fig 4). Patient 2 had cor pulmonale secondary to both restrictive and obstructive ventilatory impairment. On admission, she
TABLE II-CHANGE IN FAT MASS CALCULATED BY ELECTROLYTE BALANCE METHOD AND PREDICTED BY ENERGY INTAKE DIFFERENCE
Balance data were started on admission and were uninterrupted for patients 3 and 6; for patient 4 balance data are given after 74 days of dieting and the two balance periods were separated by a period of weight maintenance on a mixed diet taken ad libitum; for patient 5 balance data are given from the 15th day since compliance was suspect before then, the two balance periods were separated by 7 days on a DEI of 2958 kcal/day. CFM refers to daily change in fat mass calculated using electrolyte balance. DEI =digestible energy intake.
485
given a 1962 kcal liquid formula diet (absorbed energy kcal), estimated to be near her energy requirements for weight maintenance. Diuretic treatment (frusemide 80 mg/day, amiloride 10 mg/day) was not changed. In the first 13 days, her body weight fell by 114 g/day, mainly because of was
1718
fluid loss. The calculated rate of fat loss was 15 g/day. On day 14, diuretic therapy was withdrawn. There was a progressive increase in weight (3450 g over the next 14 days, 246 g/day) due to fluid retention but no significant change in the rate of fat loss (18 g/day). Calorie restriction (789 kcal/day, absorbed energy 668 kcal/day) was started on day 28, and fat and fluid loss began immediately. The calculated rate of fat loss increased over the next 14 days by 114 g/day, close to the predicted increase of 108 g/day allowing for the measured increase in daily protein loss (10’6g) and for energy losses in faeces and urine. Owing to a period of spontaneous fluid retention after day 42, the rate of weight loss decreased slowly from 264±47 g/day (days 28-41) to 111±39 g/day (days 47-60); the rate of fat loss was 110±26 g/day over this period. On day 62 frusemide (40 mg/day) was restarted; the rate of weight loss increased to 278 g/day (days 63-90) with an immediate sodium diuresis and no significant change in the rate of fat loss (139±39 g/day, days 63-90), the mean increase of 29 g/day being partly accounted for by the further reduction in the absorbed dietary energy by 96 kcal/day. The frusemide dose was reduced to 20 mg/day on day 68. Direct Measurement The tritiated
of Change in
water
space
was
Total Body Water measured twice in fourteen
patients studied between days 26 and 107 (mean weight loss 11. 50±181 kg). The measured change in total body water and the calculated change from electrolyte balance measurements were closely correlated (r= 0-922, p<0.001) and the mean loss of water estimated by the two methods (1-30±0-62v 1.55±0.65 kg) did not differ significantly.
Changes in Fat on Two Calorie Intakes Energy balance (assuming no change in total energy expenditure) was studied during two different calorie intakes in four patients (table II). In patient 3 the calorie intake was changed to induce weight loss after a period of maintenance. Patient 4 was given a calorie intake estimated to achieve weight maintenance after a period of weight loss. In two underweight patients (5 and 6, with anorexia nervosa), calorie intakes estimated to achieve weight maintenance were increased to produce weight gain. The change in fat mass calculated by our method was not significantly different from that predicted by the known change in digestible energy5 (116±15 g/day CFM versus 117±18 g/day predicted). Rate of Fat Loss from REE The rate of fat loss was estimated by multiplying the mean REE after the first 10 days of dieting by 1’. 3223 to obtain the metabolic energy deficit after subtraction of the absorbed energy intake and dividing by 9 -3kcal/g after allowing for changes in protein balance. In our seventeen patients the rate of fat loss calculated in this way correlated significantly with that calculated by the electrolyte balance method (r = 0.67, p<0. 003). The energy content of the fat loss calculated by the electrolyte balance method between days 5 and 28 was estimated to be 10.3±0.5(13) (range 6-4-13-2) kcal/g, similar to the heat of combustion of mixed fats in a bomb calorimeter (9 - 31 kcal/g").
Discussion The calculation of separate intracellular and extracellular fluid balances from electrolyte balance has been used to measure body compositional changes24 but the results may not agree with the energy changes observed. Out method, which estimates fluid changes as a single compartment, shows that patients complying with metabolic balance have constant rates of fat loss during long periods of study; we can therefore assume that the method of measuring fluid balance is reasonably precise. Even if there were a consistent unidirectional error in the fluid balance changes the rate of fat loss would still be constant, but the error would amount to that predicted by the osmotic relation 15-4 mmol sodium plus potassium for every 100 g fluid per day. The limits on the accuracy of electrolyte balance are set by the estimates used for integumental electrolyte loss, and variations in diet preparation and consumption, and collection of excreta. We have measured total exchangeable potassium twice between days 71 and 168 in six patients on metabolic balance; our methods underestimated exchangeable potassium loss by only 0 - 7±0 . 6 mmol/day (unpublished), confirming that our estimate for unmeasured potassium losses was reasonable. Our figure of 2-37 mmol potassium excreted per 1.0 g nitrogen lost is similar to the reported values from whole body counting (1.8 mmol)25 and in lean muscle tissue (2-5-3-5mmol).26 Changes in total exchangeable sodium were not measured. The variability in each individual’s daily rate of fat loss is largely a result of the variation in individual daily weight loss, which may be due to incomplete bladder emptying before weighing or variations in the contents of the gastrointestinal tract. This error can be minimised by using balance periods of at least 21 days. We have shown that the rate of fat loss is altered only by factors that change the metabolic energy deficit and is not influenced by large changes in fluid balance. Studies in obesity rarely provide objective evidence of dietary compliance during calorie restriction; metabolic balance studies, however, detect all changes in the dietary intake other than electrolyte-free carbohydrate, fat, or alcohol. Consistent additions to the prescribed diet of electrolyte-free carbohydrates can be detected in time by a fall in urinary ketone excretion. Nitrogen balance measurement is so consistent that erratic changes after adaptation to calorie restriction are usually a reliable indicator of poor compliance. If creatinine excretion is less than 75% of the individual’s weekly mean, urine collections can be assumed to be incomplete. Our method has also proved useful for patients with anorexia nervosa. The stable rate of fat loss even after long periods of continuous calorie restriction suggests that total energy expenditure remains unchanged despite reductions in both lean body mass and REE.27-29 The greater mobility after massive weight loss may explain the constant rate of fat loss occurring with a simultaneous reduction in lean body mass. The very high and rapidly declining rate of weight loss of the first 4 days was due mainly to loss of sodium and glycogen with associated water. The steady reduction in rate of weight loss in the first 28 days is due mostly to declining rates of both fluid and nitrogen loss. Since nitrogen loss depends partly on lean body mass, energy expenditure, and extent of calorie restriction (unpublished), the time for the rate of weight loss to stabilise in individual patients will be strongly influenced by these factors. The small changes in sodium balance suggest that extracellular fluid loss is negligible; the small apparent sodium retention in men probably reflects our
486
underestimate of sweat losses. A similar pattern is seen in total starvation after the initial sodium diuresis, where sodium loss is negligible30 and intracellular water loss can be predicted from urinary nitrogen.31 The small fluid loss we measured is therefore largely intracellular and is less than the 18’ 75 g intracellular water per g nitrogen of lean-tissue. This suggests either an underestimate of electrolyte losses (by about I - 5 mmol/day), or a significant contribution to the measured nitrogen loss from extracellular sites such as collagen, or both. This method of assessing changes in body fat requires no complex equipment and allows continuous monitoring of patients with a minimum of specialist attention. It can be used in outpatients with modifications for economy-food tables, urinary urea estimations, and a single week’s stool collections. It can help demonstrate to a patient without the need for repeated invasive or uncomfortable procedures that underlying fat loss is continuing, even during periods of fluid retention, often precipitated by the inappropriate use of diuretics. This knowledge often improves morale, particularly in patients whose rate of weight loss is inappropriate to the supplied energy. Since it requires daily attendance for weight measurements, urine and stool collections, and diet supplies, it combines group participation with elements of behaviour modification, helping to encourage perseverance with the diet, one of the most important requirements for successful weight loss.
PRENATAL DIAGNOSIS OF COCKAYNE’S SYNDROME A. R. LEHMANN Medical Research Council Cell Mutation
ANNE J. FRANCIS
London SE1 9RT
Cockayne’s syndrome (CS) was diagnosed prenatally by examination of amniotic cells cultured in vitro. RNA synthesis after irradiation with ultraviolet light was abnormal in cells from a fetus with CS
Summary
but
2.
3. 4. 5. 6. 7.
8. 9.
10. 11
92-113.
12 13.
14 15.
16.
17.
Lovelady HG, Stork EJ. An improved method for preparation of feces for bomb calorimetry Clin Chem 1970; 16: 253-54. Wynn V. The osmotic behaviour of the body cells in man. Significance of changes in plasma. Lancet 1957; ii 1212-18. Robinson JR. Metabolism of intracellular water. Physiol Rev 1960; 40: 112-49. Edelman IS, Leibman J, O’Meara MP, Birkenfield LW Interrelations between serum sodium concentration, serum osmolality and total exchangeable sodium, total exchangeable potassium and total body water. J Clin Invest 1958; 37: 1236-56 Vaughan BE, Boling EA. Rapid assay procedure for tritium-labelled water in body fluids. J Lab Clin Med 1961; 57: 159-64. Atwater WO, Bryant AP. Storrs (Conn). Agriculture Experimental Station Report, 1899: 73.
18.
19.
Flatt JP. The biochemistry of energy expenditure. In: Bray GA, ed. Recent advances in obesity research: II; Proceedings of the 2nd International Congress on Obesity. London Newman Publishing London, 1978: 211-28 Holmes CW, Christensen R, Carr JR, Pearson G. Some aspects of the energy metabolism of growing pigs fed on diets containing different concentrations of protein. In Mount LE, ed. Energy metabolism European Association for Animal Production publication no 26. London: Butterworth, 1980: 97-100.
not in
procedure two cases
is
cells from simple and
(one positive,
fetus which was normal. The rapid and the outcome of the test in a
one
negative) was unambiguous.
Introduction
Cockayne’s syndrome (CS) is a rare autosomal recessive characterised by cachectic dwarfism, mental retardation, progressive neurological and retinal degeneration, and skin photosensitivity.’ The recurrence risk is 1 in 4, as for all autosomal recessive disorders, and genetic counselling should be offered to parents of affected children. A prenatal diagnostic test would allow more positive and helpful advice to be given. Cultured skin fibroblasts from CS patients are hypersensitive to the lethal effects of ultraviolet (UV) light and some chemical carcinogens.2We have demonstrated that sensitivity to UV light was associated with an anomalous response of nucleic-acid synthesis in cultured CS fibroblasts after UV irradiation. Damage produced in DNA in cells from normal individuals by ultraviolet light depresses rates of both DNA and RNA synthesis but soon disorder
REFERENCES
Keys A, Brozek J, Henschel A, Mickelsen O, Taylor HL. The biology of human starvation. Minneapolis: University of Minnesota Press, 1950. Forbes CB, Drenick EJ. Loss of body nitrogen on fasting Am J Clin Nutr 1979; 32: 1570-74. Bray GA. The myth of diet in the management of obesity. Am JClin Nutr 1970; 23: 1141-48 Apfelbaum M, Bostsarron J, Lacatis D. Effect of caloric restriction and excessive caloric intake in energy expenditure. Am J Clin Nutr 1971, 24: 1405-09. Bjorntorp P, Sjostrom L. Carbohydrate storage in man: speculations and some quantitative considerations. Metabolism 1978; 27: 1853-65. Metropolitan Life Insurance Co New weight standards for men and women. Statist Bull Metropol Life Insur Co 1959; 40: 1. Gitelman HJ, Lutwak L Dermal losses of minerals in elderly women under nonsweating conditions Clin Res 1963; 11: 42 (abstr). Forbes GB. Unmeasured losses of potassium in balance studies. Am J Clin Nutr 1983; 38: 347-48. Schwartz IL. Extrarenal regulation with special reference to the sweat glands. In: Comar CL, Bronner F, eds. Mineral metabolism, vol 1 New York: Academic Press, 1960: 338-86 Aban Odduye E, Margen S. Nitrogen balance studies in humans: long-term effect of high nitrogen intake on nitrogen accretion J Nutr 1979; 10: 363-77. Ashley BCE, Whyte HM. Metabolic studies in starvation. Aust Ann Med 1961; 10:
F. GIANNELLI
Guy’s Hospital Medical School, Paediatric Research Unit,
We thank Dr M. Davie, Dr D. Evans, Dr P. Giangrande, Dr M. Kamm, Dr C. Kennedy, Dr T. Lockington, Dr J. Parr, Dr. H. Thomas, Dr. P. Thomas, Dr H. Scotland, Dr J. Smith, and Dr G. Watts, for their clinical contributions; Dr P. Baron, Mr 1. Godsland, Miss B. Hewins, Miss K. Ozin, Miss R. Ward, Mr M. Williams, and Mr S. A. Suddle, for technical assistance; Mrs Ruth Simpson, for computing help; Miss Alison McTaggart, nutritionist; Mrs Jane Biden, diet cook; and the nursing staff on the metabolic ward. Correspondence should be addressed to V. W.
1.
Unit,
University of Sussex, Brighton
after low UV doses these rates become normal. In all CS cells tested, this recovery did not occur. 3,4 The differential response of RNA synthesis in normal and CS cells after UVirradiation forms the basis of our prenatal test, which is rapid and simple. RNA synthesis is assessed by the incorporation of into amniotic cells. This a pulse-label of 3H-uridine been measured both by liquid scintillation has incorporation .
counting
22. 23.
24.
25.
26. 27 28.
29
30. 31
by autoradiography.
NJH, Denison DM. The measurement of metabolic gas exchange and minute volume by mass spectrometry alone. Resp Physiol 1979; 36: 261-67. Weir JB de V. New methods for calculating metabolic rate with special reference to protein metabolism. J Physiol 1949; 109: 1-9. Pace N, Rathbun EN. Studies on body composition: water and chemically combined nitrogen content in relation to fat content. J Biol Chem 1945; 158: 685-91. Ravussin E, Burnand B, Schutz Y, Jequier E. Twenty-four-hour energy expenditure and resting metabolic rate in obese, moderately obese, and control subjects. AmJ Clin Nutr 1982; 35: 566-73. Reifenstein EC Jr, Albright F, Wells SL The accumulation, interpretation, and presentation of data pertaining to metabolic balances, notably those of calcium, phosphorus, and nitrogen. J Clin Endocrinol 1945; 5: 367-95. Burkinshaw L, Morgan DB, Silverton NP, Thomas RD. Total body nitrogen and its relation to body potassium and fat-free mass in healthy subjects. Clin Sci 1981, 61: 457-62. Hastings AB. The electrolytes of tissues and body fluids. Harvey Lect 1941; 36: 91 Webb P, Abrams T. Loss of fat stores and reduction in sedentary energy expenditure from undereating. Hum Nutr Clin Nutr 1983; 37C: 271-82. Brown MR, Klish WJ, Hollander J, Campbell MA, Forbes GB A high protein, low calorie liquid diet in the treatment of very obese adolescents: long-term effect on lean body mass. Am J Clin Nutr 1983; 38: 20-31. Dore C, Hesp R, Wilkins D, Garrow JS Prediction of energy requirements of obese patients after massive weight loss. Hum Nutr Clin Nutr 1982; 36C: 41-48. Rapoport A, From GLA, Hudsan H. Metabolic studies in prolonged fasting. I: Inorganic metabolism and kidney function. Metabolism 1965; 14: 31-46 Drenick EJ, Swenseid ME, Blahd EH, Tuttle SG. Prolonged starvation as treatment for severe obesity. JAMA 1964; 187: 100-05.
20. Davies 21.
and
R. R. ABRAHAM
metabolic balance measurements, has enabled us to interpret the daily weight-loss pattern of patients, to become rapidly alerted to non-compliance, and to compare different dietary regimens over long periods. We have studied twenty-five obese patients during calorie restriction for up to 120 days.
J. W. DENSEM Alexander Simpson Laboratory for Metabolic Research, St Mary’s Hospital Medical School, London W2
Rates of weight and fat loss in sixteen female and nine male obese patients during calorie restriction (655-789 kcal/day) for up to 120 days were studied by a method for estimating daily changes in body composition. Fat mass is calculated by subtracting daily fluid (calculated from sodium and potassium balances) and protein mass changes from daily weight changes. After the natriuresis of the first 4 days, there was a slower rate of weight loss between days 5 and 28 due largely to a decreasing contribution from fluid and protein losses. No significant change in the rate of weight loss could be shown after the first 28 days. The rate of fat loss did not change significantly from day 5 onwards suggesting no significant change in total energy expenditure during the study period. After 68 days, 93·4% of the weight loss was fat.
Summary
Introduction THE difficulty in losing. weight reported by many obese patients after a few weeks on calorie-restricted diets may be the result of various adaptive mechanisms, such as reduced fluid and protein losses and metabolic rates as well as dietary non-compliance and fluid retention. To assess the role of these responses the composition of the weight lost during dieting must be known. Changes in fluid balance and body protein must be accounted for before ascribing weight loss to
fat. We have developed a method for the measurement of the main components of weight change during dieting. Labile carbohydrate stores (glycogen) are small and are depleted within a few days when energy intake is reduced,I,5 making no further contribution to weight loss. Changes in body water are estimated from the sum of external sodium and potassium balances and protein changes from nitrogen balance. From these data, it is possible to estimate the daily fat loss. Other indirect methods of measuring body fat are invasive, complicated, or not suitable for continuous monitoring of the patient. Our method, using strict dietary controls and
7. Hirata
Y, Ishizu H. Elevated insulin-binding capacity of serum proteins in a case of spontaneous hypoglycemia and mild diabetes not treated with insulin. Tokohu J Exp Med 1972; 107: 277-86. 8. Foiling I, Norman N. Hyperglycemia, hypoglycemic attacks and production of antiinsulin antibodies without previous known immunisation. Diabetes 1972, 21: 814-26. 9. Goldman J, Baldwin D, Rubenstein AH et al. Characterisation of circulating insulin and pro-insulin binding antibodies in autoimmune hypoglycemia. J Clin Invest 1979; 63: 1050-59. 10. Palmer JP, Asplin CM, Clemons P, et al. Insulin antibodies in insulin-dependent diabetics before insulin treatment. Science 1983; 222: 1337-39. 11. Wilkin TJ, Nicholson S. Autoantibodies against human insulin. Br Med J 1984; 288: 349-52. 12. Reeves WG. Insulin antibody determination: Theoretical and practical considerations. Diabetologia 1983; 24: 399-403. 13. Spencer KM, Tarn A, Dean BM, Lister J, Bottazzo GF. Fluctuating islet-cell autoimmunity in unaffected relatives of patients with insulin-dependent diabetes. Lancet 1984; i. 764-66. 14. Wilkin TJ, Swanson Beck J, Gunn A, Newton RW, Isles TE, Crookes J. Autoantibodies in thyrotoxicosis. A quantitative study of their behaviour in relation to the course and outcome of treatment. J Endocrinol Invest 1980; 3: 5-14. 15. Wright R Immunology of gastrointestinal and liver disease London: Edward Arnold, 1977: 16-26.
Patients and Methods Patients The mean age of the nine male patients was 38 - 6-t4 -years and their mean weight was 137-7±9-33 kg (196±16% of ideal6). The mean age of the sixteen women was 38 - 2±4 - 0 years and their mean weight was 98 - 9±4 - 8 kg (178±9% ofidear). All were clinically and biochemically euthyroid. Patients who had lost weight before admission or were on diuretic or thyroid therapy were excluded. After fasting overnight and emptying the bladder, each patient, wearing a gown, was weighed on an electronic balance with a precision of <10 g in 100 kg (coefficient of variation 0-0121%). Patients weighing more than 140 kg were weighed on a beam balance with manual zeroing and a precision of 28 g in 120 kg (coefficient of variation 0-024%). Patients did not take strenuous physical exercise but their activities on the ward were not restricted.
Electrolyte and Nitrogen Balance Patients were studied in the metabolic ward with rigorous attention to dietary intake and 24 h urine and stool collections. Lowcalorie liquid formula and mixed diets ranging between 655 and 789 kcal energy content (50-53 g protein, 37-58 mmol sodium, 49-66 mmol potassium, 30-87 g carbohydrate, 11-37 g fat, and 2-31 g fibre) were prepared from bulk supplies by a consistent technique; the composition will be reported elsewhere. Diets for weight maintenance or weight gain were prepared using ’Nutrauxil’ (Kabivitrum, Uxbridge), ’Prosparol’ (50:50 arachis oil in water, Duncan Flockhart, London), and ’Polycal’ (maltodextrins, Nutricia, The Netherlands). Vitamin and iron supplements were provided, with appropriate sodium and potassium chloride additions. The diets were analysed in triplicate monthly. Drinking water was deionised. Dietary energy content was measured by adiabatic bomb calorimetry. Flame photometry and the Kjeldahl method were used to measure the daily excretion of electrolytes and nitrogen in urine and stool samples. The latter were stored in weekly batches for analysis. The between-assay coefficients of variation were less than 3-9/0. An allowance for blood samples taken and heparinised saline infused during diagnostic tests was made for each patient. We used currently accepted figures for integumental and sweat losses (3 mmol/day sodium,2mmol/day potassium,8 and O.3 g/day nitrogen9>o), confirmed as reasonable estimates by our own measurements of sweat loss on a separate group of eight patients by the method of Ashley and Whyte." The total mean daily unmeasured losses (integumental and venesection) ranged between 5 - 5 and 9 - 3 mmol for sodium plus potassium and were 0 - 78±0’ 03 g/day for nitrogen. Mean faecal energy losses, measured by bomb calorimetry, 12 were 58 - 3±7 - 8 kcal/day nearly 9% (range, 7-12%) of the energy intake.
Assessment
of Components of Weight Loss
Body water. -Electrolytes constitute about 98% of the osmotically active solutes in body fluids.13,14 Since extracellular and intracellular fluids are in osmotic equilibrium, changes in electrolyte balance can be taken to reflect changes in body water provided extracellular osmolality remains constant.13 Total body water is closely correlated with body sodium and potassium (measured by isotope dilution) both in health and disease. We used the data of Edelman et al 15 to obtain the sum of sodium plus potassium associated with a litre of body water (153-6±5-66 mmol). Direct measurements of the change in total body water were obtained in fourteen patients by tritiated water dilution with rapid vacuum sublimation’-6 of urine, blood, and saliva.
483 TABLE 1-MEAN+SEM METABOLIC BALANCE DATA
For differences between 5-28 days and 28-68 days: *p<0.001; p<0.005. $In the ten patients studied for longer than 68 days, these percentages for day 68 to end of diet were: fat 93 - 4%, fluid I - 6%, protein 5 - 0%.
Fig 1-Mean rates of weight and fat loss. 4-day means are shown.
Body protein changes were calculated from using a factor of 6-25.
the
net
nitrogen
balance
Body fat changes were calculated by subtracting from the weight changes the body water and protein changes. Predicted Change in Absorbed Energy
Daily Fat Mass from Known Change in
Assuming constant daily energy expenditure on two diets of different energy content, the predicted change in daily fat mass was calculated by dividing the difference in the absorbed energy of the two diets by 9-3 3 kcal/g, the heat of combustion of animal fat. 17 Energy lost as protein was subtracted (using 5 - 65 kcal/g as the heat of combustion of muscle protein 17). Energy equivalents of 10-66 kcal/g and 12 kcal/g for fat and protein, respectively, were used when there was net storage. 18,199 Resting energy expenditure (REE) was measured by indirect calorimetry with a face mask. Argon dilution was used to measure minute volume and gas concentrations were measured with an MSR2 Medishield mass spectrometer. 20 The REE was calculated from the modification introduced by Weir 21 using the non-protein
day 28, the cumulative mean weight loss was linear (r = 0 - 99969, p<0. 001) (table I). For days 5 to 68, the cumulative mean rate of fat loss was linear (r = 0 99897, p<0.001; fig 1); the rate was higher in men than in women (p<0 . table 001; t). For twelve of thirteen 120 studied for to subjects up days, there was no further in rate of weight or fat loss. significant change An example of the use of our method to calculate the rate of fat loss is shown for a male patient in fig 2. The rate of fat, loss (298±18 g/day) was 90% of the rate of weight loss (330±19 g/day) and did not change significantly with time. When weight loss is not complicated by large changes in fluid balance, the rate of loss of fat becomes more closely related to the rate of weight loss as protein loss approaches an equilibrium during long periods of calorie restriction.
respiratory quotient. Statistics All results are expressed as the mean with the standard Student’s t test was used for comparisons.
error.
Results All the patients were on continuous calorie restriction for at least 28 days. Six men and seven women continued dieting for at least 68 days. In both men and women, there was an initial rapid weight loss (men 735± 102 g/day, women 477±71 g/day for days 1-4) which was significantly greater than that during days 5-8 (men 466±57 g/day, p<0.05; women 273±23 g/day, p<0.01) (fig 1). Between days 5 and 28 there was a significant exponential decrease in the daily weight loss in both groups (by 1 - 60% of the daily rate of weight loss, p<0-05); by days 25-28, the rate of weight loss was 334±33 g/day in men and 195±30 g/day in women (p<0 . 05). After
Fig 2-Cumulative weight and calculated fat loss and sodium, potassium, and nitrogen balances of patient 1 (male, aged 49 years, height 180 cm, 235% ideal body weight.)
484
Fig 4-Metabolic balance study of patient 2 (female, aged height 139-0 cm, 187% ideal body weight).
Fig 3-Daily fluid, sodium, potassium,
and
nitrogen balance.
Fluid loss in men and women was most closely associated with sodium loss in the first week, especially on the first day (fig 3). Between days 5 and 28 there was a significant fall in the rate of fluid loss by 6 - 77 g/day (p<0 . 00 I) in men and 1.93 in the mean fluid loss over this time g/day (p<0. 0 1) women, in men in than women (p<0. 01, being significantly greater table I). The fluid loss during days 5-28 is mainly a result of the continuing intracellular potassium losses that accompany protein loss and its amount and duration are greater in men. Nitrogen loss was consistently greater in men than in women (p<0’001, table I); neither group achieved zero nitrogen balance which decreased by 0-19 g/day in men and 0’ 10 g/day in women between days 5-28. Potassium loss in men and women was related to the continuing nitrogen deficit (2’337mmol potassium/g nitrogen). Until the rate of weight loss stabilised after about 28 days of calorie restriction an increasing percentage of fat and a
50 years,
of water and protein were lost (table r). The percentage of fat in the weight lost after stabilisation estimated by our method (87-93%) agreed closely with the 92 - 5% obtained from total body water measurements (assuming 73-2% water in the fat-free mass 22) in seven patients studied between 61 and 114 days of continuous calorie restriction with our diets; mean weight loss was
reducing percentage
15-02
kg.
Influence of Diuretics and Fluid Balance Changes on Calculated Rate of Fat Loss The ability to distinguish between weight loss due to changes in fluid balance and that due to-fat loss is crucial to the use of our method. The failure of large changes of fluid balance to influence substantially the cumulative fat loss slope is illustrated by an individiaul metabolic balance study (fig 4). Patient 2 had cor pulmonale secondary to both restrictive and obstructive ventilatory impairment. On admission, she
TABLE II-CHANGE IN FAT MASS CALCULATED BY ELECTROLYTE BALANCE METHOD AND PREDICTED BY ENERGY INTAKE DIFFERENCE
Balance data were started on admission and were uninterrupted for patients 3 and 6; for patient 4 balance data are given after 74 days of dieting and the two balance periods were separated by a period of weight maintenance on a mixed diet taken ad libitum; for patient 5 balance data are given from the 15th day since compliance was suspect before then, the two balance periods were separated by 7 days on a DEI of 2958 kcal/day. CFM refers to daily change in fat mass calculated using electrolyte balance. DEI =digestible energy intake.
485
given a 1962 kcal liquid formula diet (absorbed energy kcal), estimated to be near her energy requirements for weight maintenance. Diuretic treatment (frusemide 80 mg/day, amiloride 10 mg/day) was not changed. In the first 13 days, her body weight fell by 114 g/day, mainly because of was
1718
fluid loss. The calculated rate of fat loss was 15 g/day. On day 14, diuretic therapy was withdrawn. There was a progressive increase in weight (3450 g over the next 14 days, 246 g/day) due to fluid retention but no significant change in the rate of fat loss (18 g/day). Calorie restriction (789 kcal/day, absorbed energy 668 kcal/day) was started on day 28, and fat and fluid loss began immediately. The calculated rate of fat loss increased over the next 14 days by 114 g/day, close to the predicted increase of 108 g/day allowing for the measured increase in daily protein loss (10’6g) and for energy losses in faeces and urine. Owing to a period of spontaneous fluid retention after day 42, the rate of weight loss decreased slowly from 264±47 g/day (days 28-41) to 111±39 g/day (days 47-60); the rate of fat loss was 110±26 g/day over this period. On day 62 frusemide (40 mg/day) was restarted; the rate of weight loss increased to 278 g/day (days 63-90) with an immediate sodium diuresis and no significant change in the rate of fat loss (139±39 g/day, days 63-90), the mean increase of 29 g/day being partly accounted for by the further reduction in the absorbed dietary energy by 96 kcal/day. The frusemide dose was reduced to 20 mg/day on day 68. Direct Measurement The tritiated
of Change in
water
space
was
Total Body Water measured twice in fourteen
patients studied between days 26 and 107 (mean weight loss 11. 50±181 kg). The measured change in total body water and the calculated change from electrolyte balance measurements were closely correlated (r= 0-922, p<0.001) and the mean loss of water estimated by the two methods (1-30±0-62v 1.55±0.65 kg) did not differ significantly.
Changes in Fat on Two Calorie Intakes Energy balance (assuming no change in total energy expenditure) was studied during two different calorie intakes in four patients (table II). In patient 3 the calorie intake was changed to induce weight loss after a period of maintenance. Patient 4 was given a calorie intake estimated to achieve weight maintenance after a period of weight loss. In two underweight patients (5 and 6, with anorexia nervosa), calorie intakes estimated to achieve weight maintenance were increased to produce weight gain. The change in fat mass calculated by our method was not significantly different from that predicted by the known change in digestible energy5 (116±15 g/day CFM versus 117±18 g/day predicted). Rate of Fat Loss from REE The rate of fat loss was estimated by multiplying the mean REE after the first 10 days of dieting by 1’. 3223 to obtain the metabolic energy deficit after subtraction of the absorbed energy intake and dividing by 9 -3kcal/g after allowing for changes in protein balance. In our seventeen patients the rate of fat loss calculated in this way correlated significantly with that calculated by the electrolyte balance method (r = 0.67, p<0. 003). The energy content of the fat loss calculated by the electrolyte balance method between days 5 and 28 was estimated to be 10.3±0.5(13) (range 6-4-13-2) kcal/g, similar to the heat of combustion of mixed fats in a bomb calorimeter (9 - 31 kcal/g").
Discussion The calculation of separate intracellular and extracellular fluid balances from electrolyte balance has been used to measure body compositional changes24 but the results may not agree with the energy changes observed. Out method, which estimates fluid changes as a single compartment, shows that patients complying with metabolic balance have constant rates of fat loss during long periods of study; we can therefore assume that the method of measuring fluid balance is reasonably precise. Even if there were a consistent unidirectional error in the fluid balance changes the rate of fat loss would still be constant, but the error would amount to that predicted by the osmotic relation 15-4 mmol sodium plus potassium for every 100 g fluid per day. The limits on the accuracy of electrolyte balance are set by the estimates used for integumental electrolyte loss, and variations in diet preparation and consumption, and collection of excreta. We have measured total exchangeable potassium twice between days 71 and 168 in six patients on metabolic balance; our methods underestimated exchangeable potassium loss by only 0 - 7±0 . 6 mmol/day (unpublished), confirming that our estimate for unmeasured potassium losses was reasonable. Our figure of 2-37 mmol potassium excreted per 1.0 g nitrogen lost is similar to the reported values from whole body counting (1.8 mmol)25 and in lean muscle tissue (2-5-3-5mmol).26 Changes in total exchangeable sodium were not measured. The variability in each individual’s daily rate of fat loss is largely a result of the variation in individual daily weight loss, which may be due to incomplete bladder emptying before weighing or variations in the contents of the gastrointestinal tract. This error can be minimised by using balance periods of at least 21 days. We have shown that the rate of fat loss is altered only by factors that change the metabolic energy deficit and is not influenced by large changes in fluid balance. Studies in obesity rarely provide objective evidence of dietary compliance during calorie restriction; metabolic balance studies, however, detect all changes in the dietary intake other than electrolyte-free carbohydrate, fat, or alcohol. Consistent additions to the prescribed diet of electrolyte-free carbohydrates can be detected in time by a fall in urinary ketone excretion. Nitrogen balance measurement is so consistent that erratic changes after adaptation to calorie restriction are usually a reliable indicator of poor compliance. If creatinine excretion is less than 75% of the individual’s weekly mean, urine collections can be assumed to be incomplete. Our method has also proved useful for patients with anorexia nervosa. The stable rate of fat loss even after long periods of continuous calorie restriction suggests that total energy expenditure remains unchanged despite reductions in both lean body mass and REE.27-29 The greater mobility after massive weight loss may explain the constant rate of fat loss occurring with a simultaneous reduction in lean body mass. The very high and rapidly declining rate of weight loss of the first 4 days was due mainly to loss of sodium and glycogen with associated water. The steady reduction in rate of weight loss in the first 28 days is due mostly to declining rates of both fluid and nitrogen loss. Since nitrogen loss depends partly on lean body mass, energy expenditure, and extent of calorie restriction (unpublished), the time for the rate of weight loss to stabilise in individual patients will be strongly influenced by these factors. The small changes in sodium balance suggest that extracellular fluid loss is negligible; the small apparent sodium retention in men probably reflects our
486
underestimate of sweat losses. A similar pattern is seen in total starvation after the initial sodium diuresis, where sodium loss is negligible30 and intracellular water loss can be predicted from urinary nitrogen.31 The small fluid loss we measured is therefore largely intracellular and is less than the 18’ 75 g intracellular water per g nitrogen of lean-tissue. This suggests either an underestimate of electrolyte losses (by about I - 5 mmol/day), or a significant contribution to the measured nitrogen loss from extracellular sites such as collagen, or both. This method of assessing changes in body fat requires no complex equipment and allows continuous monitoring of patients with a minimum of specialist attention. It can be used in outpatients with modifications for economy-food tables, urinary urea estimations, and a single week’s stool collections. It can help demonstrate to a patient without the need for repeated invasive or uncomfortable procedures that underlying fat loss is continuing, even during periods of fluid retention, often precipitated by the inappropriate use of diuretics. This knowledge often improves morale, particularly in patients whose rate of weight loss is inappropriate to the supplied energy. Since it requires daily attendance for weight measurements, urine and stool collections, and diet supplies, it combines group participation with elements of behaviour modification, helping to encourage perseverance with the diet, one of the most important requirements for successful weight loss.
PRENATAL DIAGNOSIS OF COCKAYNE’S SYNDROME A. R. LEHMANN Medical Research Council Cell Mutation
ANNE J. FRANCIS
London SE1 9RT
Cockayne’s syndrome (CS) was diagnosed prenatally by examination of amniotic cells cultured in vitro. RNA synthesis after irradiation with ultraviolet light was abnormal in cells from a fetus with CS
Summary
but
2.
3. 4. 5. 6. 7.
8. 9.
10. 11
92-113.
12 13.
14 15.
16.
17.
Lovelady HG, Stork EJ. An improved method for preparation of feces for bomb calorimetry Clin Chem 1970; 16: 253-54. Wynn V. The osmotic behaviour of the body cells in man. Significance of changes in plasma. Lancet 1957; ii 1212-18. Robinson JR. Metabolism of intracellular water. Physiol Rev 1960; 40: 112-49. Edelman IS, Leibman J, O’Meara MP, Birkenfield LW Interrelations between serum sodium concentration, serum osmolality and total exchangeable sodium, total exchangeable potassium and total body water. J Clin Invest 1958; 37: 1236-56 Vaughan BE, Boling EA. Rapid assay procedure for tritium-labelled water in body fluids. J Lab Clin Med 1961; 57: 159-64. Atwater WO, Bryant AP. Storrs (Conn). Agriculture Experimental Station Report, 1899: 73.
18.
19.
Flatt JP. The biochemistry of energy expenditure. In: Bray GA, ed. Recent advances in obesity research: II; Proceedings of the 2nd International Congress on Obesity. London Newman Publishing London, 1978: 211-28 Holmes CW, Christensen R, Carr JR, Pearson G. Some aspects of the energy metabolism of growing pigs fed on diets containing different concentrations of protein. In Mount LE, ed. Energy metabolism European Association for Animal Production publication no 26. London: Butterworth, 1980: 97-100.
not in
procedure two cases
is
cells from simple and
(one positive,
fetus which was normal. The rapid and the outcome of the test in a
one
negative) was unambiguous.
Introduction
Cockayne’s syndrome (CS) is a rare autosomal recessive characterised by cachectic dwarfism, mental retardation, progressive neurological and retinal degeneration, and skin photosensitivity.’ The recurrence risk is 1 in 4, as for all autosomal recessive disorders, and genetic counselling should be offered to parents of affected children. A prenatal diagnostic test would allow more positive and helpful advice to be given. Cultured skin fibroblasts from CS patients are hypersensitive to the lethal effects of ultraviolet (UV) light and some chemical carcinogens.2We have demonstrated that sensitivity to UV light was associated with an anomalous response of nucleic-acid synthesis in cultured CS fibroblasts after UV irradiation. Damage produced in DNA in cells from normal individuals by ultraviolet light depresses rates of both DNA and RNA synthesis but soon disorder
REFERENCES
Keys A, Brozek J, Henschel A, Mickelsen O, Taylor HL. The biology of human starvation. Minneapolis: University of Minnesota Press, 1950. Forbes CB, Drenick EJ. Loss of body nitrogen on fasting Am J Clin Nutr 1979; 32: 1570-74. Bray GA. The myth of diet in the management of obesity. Am JClin Nutr 1970; 23: 1141-48 Apfelbaum M, Bostsarron J, Lacatis D. Effect of caloric restriction and excessive caloric intake in energy expenditure. Am J Clin Nutr 1971, 24: 1405-09. Bjorntorp P, Sjostrom L. Carbohydrate storage in man: speculations and some quantitative considerations. Metabolism 1978; 27: 1853-65. Metropolitan Life Insurance Co New weight standards for men and women. Statist Bull Metropol Life Insur Co 1959; 40: 1. Gitelman HJ, Lutwak L Dermal losses of minerals in elderly women under nonsweating conditions Clin Res 1963; 11: 42 (abstr). Forbes GB. Unmeasured losses of potassium in balance studies. Am J Clin Nutr 1983; 38: 347-48. Schwartz IL. Extrarenal regulation with special reference to the sweat glands. In: Comar CL, Bronner F, eds. Mineral metabolism, vol 1 New York: Academic Press, 1960: 338-86 Aban Odduye E, Margen S. Nitrogen balance studies in humans: long-term effect of high nitrogen intake on nitrogen accretion J Nutr 1979; 10: 363-77. Ashley BCE, Whyte HM. Metabolic studies in starvation. Aust Ann Med 1961; 10:
F. GIANNELLI
Guy’s Hospital Medical School, Paediatric Research Unit,
We thank Dr M. Davie, Dr D. Evans, Dr P. Giangrande, Dr M. Kamm, Dr C. Kennedy, Dr T. Lockington, Dr J. Parr, Dr. H. Thomas, Dr. P. Thomas, Dr H. Scotland, Dr J. Smith, and Dr G. Watts, for their clinical contributions; Dr P. Baron, Mr 1. Godsland, Miss B. Hewins, Miss K. Ozin, Miss R. Ward, Mr M. Williams, and Mr S. A. Suddle, for technical assistance; Mrs Ruth Simpson, for computing help; Miss Alison McTaggart, nutritionist; Mrs Jane Biden, diet cook; and the nursing staff on the metabolic ward. Correspondence should be addressed to V. W.
1.
Unit,
University of Sussex, Brighton
after low UV doses these rates become normal. In all CS cells tested, this recovery did not occur. 3,4 The differential response of RNA synthesis in normal and CS cells after UVirradiation forms the basis of our prenatal test, which is rapid and simple. RNA synthesis is assessed by the incorporation of into amniotic cells. This a pulse-label of 3H-uridine been measured both by liquid scintillation has incorporation .
counting
22. 23.
24.
25.
26. 27 28.
29
30. 31
by autoradiography.
NJH, Denison DM. The measurement of metabolic gas exchange and minute volume by mass spectrometry alone. Resp Physiol 1979; 36: 261-67. Weir JB de V. New methods for calculating metabolic rate with special reference to protein metabolism. J Physiol 1949; 109: 1-9. Pace N, Rathbun EN. Studies on body composition: water and chemically combined nitrogen content in relation to fat content. J Biol Chem 1945; 158: 685-91. Ravussin E, Burnand B, Schutz Y, Jequier E. Twenty-four-hour energy expenditure and resting metabolic rate in obese, moderately obese, and control subjects. AmJ Clin Nutr 1982; 35: 566-73. Reifenstein EC Jr, Albright F, Wells SL The accumulation, interpretation, and presentation of data pertaining to metabolic balances, notably those of calcium, phosphorus, and nitrogen. J Clin Endocrinol 1945; 5: 367-95. Burkinshaw L, Morgan DB, Silverton NP, Thomas RD. Total body nitrogen and its relation to body potassium and fat-free mass in healthy subjects. Clin Sci 1981, 61: 457-62. Hastings AB. The electrolytes of tissues and body fluids. Harvey Lect 1941; 36: 91 Webb P, Abrams T. Loss of fat stores and reduction in sedentary energy expenditure from undereating. Hum Nutr Clin Nutr 1983; 37C: 271-82. Brown MR, Klish WJ, Hollander J, Campbell MA, Forbes GB A high protein, low calorie liquid diet in the treatment of very obese adolescents: long-term effect on lean body mass. Am J Clin Nutr 1983; 38: 20-31. Dore C, Hesp R, Wilkins D, Garrow JS Prediction of energy requirements of obese patients after massive weight loss. Hum Nutr Clin Nutr 1982; 36C: 41-48. Rapoport A, From GLA, Hudsan H. Metabolic studies in prolonged fasting. I: Inorganic metabolism and kidney function. Metabolism 1965; 14: 31-46 Drenick EJ, Swenseid ME, Blahd EH, Tuttle SG. Prolonged starvation as treatment for severe obesity. JAMA 1964; 187: 100-05.
20. Davies 21.
and