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EFFECT OF HYDRAZINE SULPHATE ON WHOLE-BODY PROTEIN BREAKDOWN MEASURED BY 14C-LYSINE METABOLISM IN LUNG CANCER PATIENTS

[Lancet 2:241-244, 1987]

JOHN A. TAYEK, DAVID HEBER, ROWAN T. CHLEBOWSKI

Department of Medicine, UCLA School of Medicine, Divisions of Endocrinology, Metabolism and Nutrition, and Medical Oncology, Harbor-UCLA Medical Center, Torrance, California, USA; and Division of Clinical Nutrition, Center of Health Sciences, Los Angeles, California

Summary

In a prospective double-blind trial twelve malnourished patients with lung cancer were randomised to receive either placebo or hydrazine sulphate (60 mg three times daily) for 30 days. Fasting lysine flux was determined by a primed 4-hour continuous infusion of 14C-lysine before and after one month of hydrazine treatment. Baseline plasma lysine flux was 2580 (SD 580) µmol/h for the placebo group and 2510 (440) µmol/h for the hydrazine group. After one month the placebo group showed a slight rise to 2920 (450) µmol/h (p=0.08) and the hydrazine group showed a significant fall to 1840 (750) µmol/h (p<0.05); serum albumin fell in the placebo group and was unchanged in the hydrazine group. Administration of hydrazine sulphate to reduce aminoacid flux may favourably influence the metabolic abnormalities in cancer cachexia.

Introduction

In patients with lung cancer, weight loss indicates poor prospects for survival. However, provision of extra calories by means of total parenteral nutrition has yielded no clinical benefit in either small-cell or non-small-cell lung cancer populations. It seems that, if more successful treatment strategies are to be developed, we need a better understanding of the mechanisms underlying cancer cachexia. Various metabolic abnormalities have been identified including increased energy expenditure and altered carbohydrate and protein metabolism. In the absence of tumour response such metabolic defects persist in lung cancer patients.

Hydrazine sulphate is an agent that in rats inhibits the enzyme phosphoenol pyruvate carboxykinase with resultant interruption in gluconeogenesis. For this reason it was proposed as a potential anti-cachexia agent and on preliminary clinical evaluation it improved abnormal glucose metabolism and weight maintenance in lung cancer patients. Hydrazine sulphate might be expected to modify whole-body protein metabolism as well. The effective inhibition of phosphoenol pyruvate carboxykinase reduces the demand for gluconeogenic precursors that might reduce amino acid releasec due to protein breakdown. We report here a short-term study of its effects on lysine flux in lung cancer patients with weight loss.

Materials and Methods

Eleven patients with non-oat-cell and one with oat-cell carcinoma were recruited over one year and were randomised to receive hydrazine sulphate 60 mg or placebo three times a day for one month. Table 1 summarises clinical nutritional characteristics and group assignment. To limit any effects of chemotherapy on measured variables, we studied patients either before therapy on measured variable or at least three weeks after their latest course. In ten patients the regimen was cisplatin 100 mg/sq m, vinblastine 4 mg/sq m, and bleomycin 10 units/sq m (CVB) once a month; patient 8 received cisplatin 50 mg/sq m, doxorubicin 50 mg/sq m, cyclophosphamide 350 mg/sq m, lomustine 50 mg/sq m, and vincristine 1-4 mg/sq m; and patient 6 (oat-cell carcinoma) received vincristine 1-4 mg/sq m, doxorubicin 50 mg/sq m, and cyclophosphamide 650 mg/sq m.

[Figure 1]

Lysine, alanine, and branched-chain-aminoacid concentrations were measured in sulposalicylic-acid-precipitated plasma samples with a Beckman 119CL autmoated aminoacid analyser. For lysine flux estimations we administered a loading dose of 2·55 µCi/L-[U-C]-lysine and then gave a constant infusion of 2 µCi/h for 4 h. The method of determining lysine flux was originally described by Waterlow and has been validated by others. Plasma C-lysine radioactivity was measured in the acid soluble fraction with a Beckman B scintillation counter as described by Heber et al. Plasma lysine specific radioactivity was measured every 20 min between hours 3 and 4 (fig 1) to ensure that steady-state conditions existed.

The lysine flux calculation is based on a conventional tracer dilution expression of plasma soluble L-[14C]-lysine enrichment during a period of isotope steady state. The lysine flux in µmol/h was calculated from the following formula: lysine flux = 14C-lysine infusate / SP, where the 14C-lysine infusate is the radioactivity infused in dpm/h and the Sp is the specific activity of 14C-lysine per µmol plasma lysine. The term flux denotes the movement of aminoacid into and out of the plasma pool. Under the steady conditions of the present study, movement into the pool equals movement out of the pool -- i.e., flux-in equals flux-out. Flux-in consists of aminoacids entering from intake, aminoacids synthesized de novo, and aminoacids entering from protein breakdown. Since the patients were fasting and lysine is an essential aminoacid, the intake and de-novo-synthesis terms are zero and aminoacid flux is a measure of lysine arising from protein breakdown. Thus these measurements of lysine flux provide an index of overall protein breakdown.

Fasting plasma glucose, insulin, thryoxine (T4), triiodothyronine (T3), free T3, and cortisol were measured in all patients. Plasma glucose was measured with a Beckman glucose analyzer. Plasma insulin, T4, T3 and cortisol were measured by standard double antibody radioimmunoassay methods. Free T3 was measured by diffusion dialysis.

For statistical evaluation we did paired comparisons for each patient. The paired t-test (two-tailed) was used to analyze differences over time in the same patient, a p value of less than 0.05 being regarded as significant. Data are expressed as mean (SD) unless otherwise stated.

Results

At baseline the placebo and hydrazine groups were similar in terms of performance scored and sex distribution (table I). Although the hydrazine group was older, age is not an important factor in estimates of protein metabolism unless there are differences in lean body mass; here lean body mass did not differ significantly between the groups, as judged by anthropometry and the percentage ideal body weight.

Table II shows the influence of hydrazine sulphate on lysine flux; at one month there was a significant reduction in the hydrazine group and a non-significant increase in the placebo group.

Table III records other changes. Plasma isoleucine declined in the placebo group; plasma alanine rose in the hydrazine group; serum albumin declined in the placebo group and was unchanged in the hydrazine group. There was a correlation between free T3 (and to a lesser extent T3) and lysine flux (fig 2). No other fasting hormone levels correlated with lysine flux. Over the one-month trial period there were no significant changes in T3, free T3, T4, or cortisol in either group.

Discussion

Lysine is the fourth most abundant aminoacid released into plasma in the fasting state, only alanine, glutamine, and glycine being greater. Alanine was raised after one month of hydrazine but not after placebo; isoleucine did not change in the hydrazine group but declined in the placebo group. Other investigators have reported lower concentrations of alanine, glutamine, and isoleucine in fasting malnourished cancer patients than in healthy controls. Effective inhibition of phosphoenol pyruvate carboxykinase should reduce the requirements for alanine and other gluconeogenic precursors. Such a reduction in demand could raise the concentration of these precursors in the plasma compartment.

Plasma leucine and valine followed the same trend as isoleucine and Tayek et al have earlier reported that the concentrations of these three aminoacids are positively correlated with albumin and whole-body protein synthesis but unrelated to whole-body protein breakdown. The decline of isoleucine in the placebo group may reflect a reduction in muscle protein synthesis, but no direct measurements were made.

The subnormal protein synthesis in skeletal muscle of cancer patients is believed to be a primary cause of the reduction in muscle mass and weight loss, and the severity of weight loss in lung carcinomas (and in several other common cancers) is a prognostic factor for survival. Cancer cachexia is associated with increases in the movement of aminoacids into and out of the plasma compartment that are believed to reflect whole-body protein flux. Increases in flux, as well as reductions in skeletal muscle protein synthesis, may be viewed as means of providing aminoacids for the synthetic requirements of visceral and tumour proteins in the malnourished patient with advancing cancer.

Muscle protein synthesis is negatively correlated with turnour burden, so aminoacids normally required by the muscle protein may not be available - hence the muscle wasting. However, it has not proved possible to replace the lost lean body mass in cancer patients with supplementary protein and energy, whether given in adequate or very large amounts. So the mechanisms are more complex than mere supply and demand. Proposed explanations include a rise in metabolic rate, and increases in both fasting aminoacid flux and glucose production.

We have earlier reported that hydrazine improves glucose tolerance and reduces fasting glucose production; here we show that it also reduces fasting lysine flux. This reduction was coupled with maintenance of serum albumin over the month of treatment, whereas in the placebo group serum albumin declined. An important predictor of 2-year survival in lung cancer patients is the maintenance of serum albumin three months from the start of chemotherapy; so the metabolic changes induced by the hydrazine group may prove clinically beneficial.

 

Table I - Clinical and Nutritional Characteristics at Baseline

- Placebo Hydrazine

Age: mean (SD), range 51 (7), 43-61 63 (10), 48-76*
Sex: F:M 5:1 5:1
Performance score 1-2 5 5
  3-4 1 1
Prior therapy None 1 3
  Chemo/radiation 5 3
Concurrent chemotherapy CVB 5 5
  Other 1 1
Usual weight (hg) 73.5 (13.8) 69.8 (10.8)
Entry weight (kg) 64.4 (16.2) 62.8 (8.5)
Ideal body weight (%) 101.7 (22.8) 101.6 (19.3)
Triceps skinfold (mm) 11.9 (11.3) 16.5 (11.4)
(percentile)** (26) (39)
Mid arm circumference (cm) 28.5 (5.3) 27.4 (4.6)
(percentile)** (37) (33)
Mid arm muscle area (cm²) 49 (14) 39 (6)
(percentile)** (51) (32)
Albumin (g/dl) 3.6 (0.3) 3.5 (0.5)
Transferrin (mg/dl) 195 (84) 176 (53)

*p < 0.05 by t test; **percentile of normal population for age and sex.

 

Table II - Whole-body Lysine Flux in Lung Cancer Patients

 
  Lysine Flux (µmol/h)
-
  Baseline 1 mo

Placebo group    
1 2172 2812
2 1869 2959
3 2373 2674
4 2772 2269
5 2758 3195
6 3542 3585

Mean (SD) 2580 (580) 2920 (450)*, **

Hydrazine treated    
7 2675 1146
8 2522 2119
9 2666 3129
10 1808 1217
11 2264 1438
12 3114 2006

Mean (SD) 2510 (440) 1840 (750)**, ***

*p = 0.08; **p < 0.05, paired t-tests with baseline
***p < 0.01 by combined paired t-test both groups

 

Table III - Fasting Biochemical and Hormonal Indices

  Placebo Hydrazine
 

- Baseline 1 mo Baseline 1 mo

Lysine (µmol/l) 149 (41) 155 (39) 140 (33) 111 (37)
Leucine (µmol/l) 101 (28) 89 (20) 98 (8) 92 (16)
Isoleucine (µmol/l) 59 (12) 49 (7)* 51 (10) 49 (9)
Valine (µmol/l) 189 (32) 171(31) 190 (26) 223 (67)
Alanine (µmol/l) 261 (73) 272 (83) 232 (53) 355 (139)*
Albumin (g/dl) 3.6 (0.3) 3.3 (0.5)** 3.5 (0.5) 3.6 (0.4)**
Transferrin (mg/dl) 195 (84) 185 (55) 176 (53) 191 (27)
T4 (µg/dl) 8.3 (1.6) 7.7 (1.6) 5.4 (1.0) 6.8 (1.6)
T3 (ng/dl) 132 (54) 157 (18) 124 (28) 158 (53)
Free T3 (pg/dl) 330 (107) 401 (47) 395 (134) 411 (87)
Glucose (mg/dl) 103 (12) 113 (30) 105 (28) 96 (17)
Insulin (µU/ml) 17 (10) 23 (13) 18 (14) 28 (31)
Cortisol (µg/dl) 21 (9) 15 (6) 25 (4) 23 (9)

*p < 0.05 by paired t-test with baseline; **p < 0.01 by combine paired t-test; *** outliers removed from analysis p < 0.01 (Dixon WJ, Biometrics 1953; 9:74-89)

 

We thank Prof Jo Anne Brasel for her valuable advice and review of this work; Stephanie Griffiths, Mario Paredes, and Maria Lajoie for technical assistance; Linda Bulcavage, RN for patient management; Mary Grosvenor, RD for nutritional evaluations; Josie Martinez for secretarial assistance; and the nurses of the Clinical Research Center for their cooperation.

This work was supported by American Cancer Society grant RD-163; NIH grants CA 37320 and CA 265631, General Clinical Research Center RR00425, Nutrition and Metabolism Training grant AM07461, and UCLA Clinical Nutrition Research Unit grant CA 42710.

Correspondence should be addressed to R. T. C., Division of Medical Oncology, Department of Medicine, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, CA 90509, USA.

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