- Email: [email protected]
Effect of growth hormone on lymphocyte respiration and growth rate of children
Effect of Growth Hormone on Lymphocyte and Growth Rate of Children G. B. Kuna, P. J. Collipp, D. Katkocin, Oxygen consumption by circulating lymphocytes of children with isolated growth hormone deficiency was studied before and 6 mo after the start of growth hormone therapy. A plot of percent change in respiration (oxygen consumed per mg protein or pg DNA) against height gain during the therapy showed a linear association (correlation coefficient 0.667-0.756).
L IVER ferential
MITOCHONDRIA isolated by difcentrifugation’ were found to accumulate 40% of growth hormone that was taken up by liver as early as 5 min after an intravenous injection into hypophysectomized rats. These results have been confirmed and it was shown’ by electron microscope autoradiography that mitochondria accumulate as much as 70% of the hormone taken up by liver. Liver mitochondria of hypophysectomized rats have several abnormalities of structure and function, such as decreased protein synthesis,’ prolonged turnover rate,4 decreased cytochrome levels and enzymes of electron transport,’ and decreased ADP-stimulated respiration.‘j Subsequent administration of growth hormone partially restored the mitochondrial activities. These studies suggest that growth hormone, in addition to other hormones, may be essential for maintenance of a normally functioning population of mitochondria of rat liver. Studies of white cell number and morphology have been used for diagnostic and therapeutic purposes. Several studies have demonstrated that white blood cells possess functioning mitoEnzymes of Krebs cycle’*” and chondria.‘,” oxidative phosphorylation” have been known to occur. Recently, Nessi et al.‘* have demonstrated ADP-stimulated respiration in mitochondria isolated from white cells. Hypothyroidism in humans has been shown to result in certain functional abnormalities of white blood cells.13 Circulating lymphocytes present a readily available tissue for biopsy. In the light of the results obtained with experimental animals,3~6~‘4 we undertook a study of the effects of growth hormone treatment on lymphocyte cell respiration in children with isolated growth hormone deficiency before and after the hormone therapy. A quantitative estimation of the mitochondrial Metabolism, Vol. 28, No. 12 (December), 1979
J. Sherwyn,
S. Amin,
Respiration
and V. T. Maddaiah
effect may be of some help in predicting the response of a given child to growth hormone replacement therapy. MATERIALS
AND
METHODS
Examination and Treatment of Children Out of 28 children that were being followed in the Growth Clinic of the Nassau County Medical Center with the diagnosis of isolated growth hormone deficiency, 12 were included in this study. Parents of these children were informed of the study and gave consent by signing the appropriate forms. Growth hormone treatment was discontinued 4 mo prior to the onset of the study in 8 subjects, and 4 of them had not previously received growth hormone. Growth hormone deficiency was diagnosed by measuring serum hormone levels at different time periods before and after insulin-induced hypoglycemia and stimulation by arginine infusion. Levels of ACTH, TSH, FSH, LH, T,. T,, and CBC were measured in children who showed growth hormone deficiency. Adrenal-pituitary axis was checked by measuring 24-hr urinary levels of 17-ketosteroids and 17ketogenic steroids before and after stimulation by metapyrone. All biochemical analyses were done at the Special Chemistry Laboratories at the Nassau County Medical Center by standard methods. All the children included in the study had normal skull x-ray and girls had normal patterns of buccal smear. X-ray of the left hand and wrist was obtained to evaluate the bone age. The children were put on hormone therapy with 2 IU of hGH (National Pituitary Agency HG-76) administered subcutaneously 3 times a wk and were on no other medication. The children were seen after 8 wk and again after 6 mo. All children were without breakfast the morning of the visit, had gone 24 hr since the last hormone injection, and were examined prior to blood drawing to detect the presence of any infection. Those with infection were excluded from the study. Height was carefully measured against a fixed wall-scale and blood was drawn by the same physician at 9 a.m. at each visit, minimizing diurnal variations.
Isolation of Lymphocytes The method is essentially that developed by Boyurn.” All flasks and syringes were rinsed with 10% EDTA. Approximately 40 ml of venous blood was drawn into a syringe rinsed
From the Department of Pediatrics, Nassau County Medical Center, State University of New York, Stony Brook Health Sciences Center. East Meadow, N. Y. Received for publication March I, 1979. Address reprint requests to V. T. Maddaiah. Ph.D., Department of Pediatrics, Nassau County Medical Center. East Meadow, N. Y. 11554. 0 I979 by Grune & Stratton. Inc. 0026-0495/79/2812-0009$01.00/0 1239
1240
KUNA ET AL.
with 10% EDTA and transferred into the flask containing 1 ml of 10% EDTA. ADP (4 mg/lO ml blood) was added and the samples were incubated at 37OC for 10 min in a rocking incubator chamber to facilitate aggregation of platelets. The blood was layered on top of a chilled mixture of 6% dextran T-500 (Pharmacia) and 39.8% sodium metrizoate solution (2~1) and allowed to stand at O°C. Separation of white cells was complete in about 90 min and all other procedures were performed at O°C. The top brownish layer containing white cells and plasma was gently pipetted out into polyethylene tubes and centrifuged at 4500 g for 10 min. The pellet at the bottom containing white cells was then resuspended by gently blowing air with a bulb-pipette and was subsequently washed three times with the modified Krebs-Ringer solution that was prepared by mixing 0.154-M solutions of NaCl, KCI, MgSO, . 7Hr0, and KHrPO, in the ratio of 9:4: 1: 1 and adjusting the pH to 7.4 with IO M NaOH. The washing procedure removed serum and erythrocytes. Any red button containing hemoglobin and RBC was carefully removed. The final white cell pellet was resuspended in the isolation medium for further use. The cells were examined microscopically using Wright’s stain, and found to be 100% lymphocytes (Fig. 1). Respiration of lymphocytes (l-2 million cells) was monitored polarographically at 30°C with a Clarke-type oxygen electrode (Yellow Springs Instrument, Yellow Springs, Ohio) fitted with a strip chart recorder (Texas Instruments,
Fig. 1.
Houston, Tex.). Incubations were carried out in a jacketed glass chamber containing the respiration medium (isolation medium containing glucose at a final concentration of 0.05%). The whole procedure was terminated by the addition of 1% potassium cyanide solution. The upper limit for 100% oxygen consumption was obtained by adding sodium dithionite powder to the respiration medium. Protein was estimated by the method described by Lowry et a1.l6 using bovine serum albumin as the standard. DNA was estimated by the mithramycin method of Hill and Whately.” Oxygen consumption was expressed as natoms and patoms of oxygen per mg protein and per rg DNA per hour, respectively. All biochemicals were from Sigma Chemical, St. Louis, MO.
RESULTS
Table 1 shows skeletal and chronologic ages of children that were included in the study. The male-female ratio was 8:4. All the children had short stature ranging from -2 to -4 SD below the mean and all had a retarded bone age and isolated growth hormone deficiency. The total white blood counts before and after 6 mo of growth hormone therapy in these children were 6778 and 7142, respectively. Differential counts
A photomicrograph of a smear of a typical preparation of lymphocytes (Wright’s stain, x 400).
GROWTH
HORMONE
ON LYMPHOCYTE
1241
RESPIRATION
Table 1. Growth Characteristics of Subjects Height Increase Height
Subwt
Age (vr)
During
Before
6 mo
Bone Age
Therapy
Therapy
Irl
(inches)
(inches)
sex
1
7
F
4
40%
2%
2
7
M
3
40%
1%
3
12
M
9
50%
1%
4
6
M
4
37%
2%
c
5
3
M
1
29%
1%
al
6
8
M
7
46%
z
2
14
7
M
12
53%
1%
P 0
8
11
F
9
48
1%
E
9
5
F
3
38%
1%
g
10
9
F
7
1%
11
11
M
10
44 42%
12
9
M
6
45%
1%
1
-100’ 1.0
00
&Its Fig. 2. Polarographic tracings of oxygen consumption by isolated lymphocytes (1.8 x 10’ cells1 in a medium (pH 7.41 containing 0.0824 M NaCI, 0.0410 M KCI. 0.0103 M MgSO, 0.0103 M KH,PO. and 0.0028 M glucose. Cells end KCN (final concentration of 0.5 mM were added as indicated.
Inches
30
i6monthsl
granulocytes, monocytes, and lymphocytes (T and B). Some of these factors may have contributed to the large variation in respiration found among the children before and after the therapy. The range of respiratory rates were 14 l-504 natoms of O,/mg protein/hr or 27-l 03 patoms of O,/pg DNA/hr before and after the hormone therapy, respectively. Since the rates varied widely, we chose to relate the percent
2
0
E
+400
-I
5
2
=
+300-
k
atoms
KCN
I”
A plot of percent change in respiration Fig. 3. expressed as natoms of oxygenlhrlmg protein (respiration after growth hormone treatment/respiration before growth hormone treatment x 100). The line is drawn for the best-fitting linear regression equation with the value of correlation coefficient (r) as shown.
.z 2 0z 5 +100P
of oxygen
20
Growth
s ‘$
I
----t t
I
3 75 minutes
I 0.36~
504
1%
were also not significantly different before and after growth hormone treatment (lymphocytes 43% and 45%, granulocytes 57% and 55%). Figure 2 shows a typical oxygen consumption curve. Oxygen consumption was increased after the addition of cells obtained from a child prior to growth hormone therapy. The increase was completely inhibited by KCN, suggesting that the increased consumption was due to mitochondrial respiration. The rate of oxygen consumption was calculated from the slopes.‘* Figure 3 shows a plot of percent change in the rate of respiration/mg protein/hr against increase in height during a period of 6 mo of growth hormone therapy. Lymphocyte respiration may be influenced by several factors, such as isolation techniques, presence of infection, phagocytosis, level of maturation, and relative composition of different types of white cells like
T
-
I 0 E z t a
+200-
. .
o-
-100
0.0
I 1.0
I
2.0
1
3.0
Growthin Inches (6 monthsI Fig. 4. A plot of percent change in respiration expressed as patoms of oxygenlhrlpg DNA (respiration after growth hormone treatment/respiration before growth hormone treatment x 100). The line is drawn for the best-fitting linear regression equation with the value of correlation coefficient (r) as shown
KUNA ET AL.
1242
change in the rate of respiration (respiration after GH treatment/rate of respiration before GH treatment x 100) to growth (increase in height). Correlation coefficient for a linear association between the two parameters (Fig. 2) was 0.667. A plot (not shown) of percent change in respiration against height gain after 8 wk of therapy gave a similar correlation. Figure 4 shows a plot of percent change in respirationlpg DNA/hr against increase in height. A value of 0.756 was obtained for the correlation coefficient for a linear association. Since contamination by DNA of cells other than white cells in the preparation is smaller than contamination by proteins, the values for slope and correlation coefficient are slightly larger for DNA data than for those based on protein. Also, percent change in respiration on the DNA basis was larger than that on a protein basis. DISCUSSION
Although circulating white cells constitute readily available nucleated cells for biochemical studies, they represent a heterogenous group of cells with different structure, function, lifespan, and source of origin.” Different cell types may have different complements of mitochondria. In whole blood, white cells as well as platelets contribute to oxygen consumption. The endogenous respiration of a white cell is approximately 45 times that of an individual platelet. The normal ratio of platelets to white cell is approximately 50: 1. As a result, the amount of oxygen consumed by platelets is equal to white blood cells in a unit volume of blood. Drugs such as thiouracil and chloramphenicol decrease white cell respiration whereas vitamin C increases it. In spite of these and other factors that could affect the rate of oxygen consumption, it is noteworthy that a fairly linear association was
observed between percent change in respiration of lymphocytes and linear growth during growth hormone therapy. Thyroxine is one of the hormones that has been found to influence the metabolic activity of white cells. Hypothyroidism decreased oxygen consumption and the rate of deiodination of thyroxine by white cells, but hyperthyroidism increased both activities.” Farid et al.13 have demonstrated by the qualitative nitroblue-tetrazolium test that polymorphonuclear leukocytes of hypothyroid patients reduced the dye less well when compared to euthyroids. The activity was restored following thyroxine treatment of hypothyroid patients. The present results suggest that growth hormone may also play a role in the regulation of respiratory activity of lymphocytes. The association of increase in respiratory activity of white cells with the increase in height of growth-hormone-deficient children indicates that the mitochondrial effect may be related to the growth-promoting property of the hormone. As discussed elsewhereI with regard to liver mitochondrial activity of experimental animals, growth hormone may influence lymphocyte mitochondrial processes such as protein synthesis, turnover, and component activities of electron transport. Whether these effects are also mediated by somatomedin is not presently known. Since percent change in respiration of lymphocytes measured only 8 wk after growth hormone therapy was also related to height gain, it may become possible to use the early respiratory increment to predict the further efficacy of therapy. ACKNOWLEDGMENT The authors thank the National Pituitary Agency (NIH), for providing human growth hormone, and Pfizer, Inc., Conn., for providing mithramycin.
REFERENCES 1. Maddaiah
VT, Rezvani 1, Chen SY. et al: Distribution of ‘H-acetyl hGH in subcellular fractions of rat liver. Biochem Med 4:492, 1970 2. Groves WE, Houts GE, Bayse GS: Subcellular distribution of ‘2sI-labeled bovine growth hormone in rat liver and kidney. Biochem Biophys Acta 264:472, 1972 3. Maddaiah VT, Sharma RK, Balachandar V, et al: Effect of GH on mitochondrial protein synthesis. J Biol Chem 248:4263,1973 4. Maddaiah VT, Collipp PJ, Lin JH, et al: Growth hormone and liver mitochondria effect on morphology and protein turnover. Biochem Med 16:47, 1976
5. Maddaiah VT, Weston CL, Chen SY, et al: GH and liver mitochondria, effects on cytochromes and some enzymes. Arch Biochem Biophys 173:225, 1976 6. Gupta K, Katkocin DM, Kostyrka ML, et al: Effect of growth hormone on rat liver mitochondria. Fed Proc 36:323, 1977 7. Jemelin M, Frei J: Leukocyte energy metabolism. III anerobic and aerobic ATP production and reiated enzymes. Enzymol Biol Clin 2:289, 1970 8. Frei J, Jemelin M, Aleyassine H: Le metabolisme energetique du leucocyte. Ann Biochem Exp Med 295, 1969
GROWTH
HORMONE
ON LYMPHOCYTE
1243
RESPIRATION
9. McKinney GR, Martin SP, Rindles RW, et al: Respiratory and glycolytic activities of human leukocytes in vitro. J Appl Physiol 5:335, 1953 10. Beck WS. Valentine WN: The carbohydrate metabolism of leukocytes: A review. Cancer Res 13:309, 1953 1 I. Evans AE, Getz GS: Mitochondrial electron transport enzymes in normal leukemic leukocytes. Blood 3 I :7 10, 1968 12. Nessi P, Billesbolle S, Fornerod M, et al: Leukocyte energy metabolism VII. Respiratory chain enzymes, oxygen consumption and oxidative phosphorylation of mitochondria isolated from leukocytes. Enzyme 22:I 83, 1977 13. Farid NR. Au B, Woodford G, et al: Polymorphonuclear leukocyte function in hypothyroidism. Hormone Res 7~247, 1976 14. Maddaiah VT, Collipp PJ: Hormonal regulation of mitochondrial growth, structure and function, in Talwar GP (ed): Regulation of Growth and Differentiated Function in Eukaryote Cells. New York, Raven, 1975, pp 453-459
IS. Boyum A: Separation of leukocytes from blood and bone marrow. Stand J Chn Lab Invest 21 (Suppl97):77-89, 1968 16. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265, 195 1 17. Hill BT, Whately S: A simple rapid DNA. FEBS Lett 56:20, 1975
microassay
for
18. Estabrook RW: Mitochondrial respiratory control and the polarographic measurement of ADP: Ratios. in Estabrook RW, Pullman ME (eds): Methods in Enzymology, vol 10. New York, Academic, 1967, pp 41-47 19. Cline MJ: Metabolism Physiol Rev 45:675, 1965
of the circulating
leukocyte.
20. Kurland GS. Krotkov MV. Freedberg AS: Oxygen consumption and thyroxine deiodination by human leukocytes. J Clin Endocrinol 20:35, 1960
L IVER ferential
MITOCHONDRIA isolated by difcentrifugation’ were found to accumulate 40% of growth hormone that was taken up by liver as early as 5 min after an intravenous injection into hypophysectomized rats. These results have been confirmed and it was shown’ by electron microscope autoradiography that mitochondria accumulate as much as 70% of the hormone taken up by liver. Liver mitochondria of hypophysectomized rats have several abnormalities of structure and function, such as decreased protein synthesis,’ prolonged turnover rate,4 decreased cytochrome levels and enzymes of electron transport,’ and decreased ADP-stimulated respiration.‘j Subsequent administration of growth hormone partially restored the mitochondrial activities. These studies suggest that growth hormone, in addition to other hormones, may be essential for maintenance of a normally functioning population of mitochondria of rat liver. Studies of white cell number and morphology have been used for diagnostic and therapeutic purposes. Several studies have demonstrated that white blood cells possess functioning mitoEnzymes of Krebs cycle’*” and chondria.‘,” oxidative phosphorylation” have been known to occur. Recently, Nessi et al.‘* have demonstrated ADP-stimulated respiration in mitochondria isolated from white cells. Hypothyroidism in humans has been shown to result in certain functional abnormalities of white blood cells.13 Circulating lymphocytes present a readily available tissue for biopsy. In the light of the results obtained with experimental animals,3~6~‘4 we undertook a study of the effects of growth hormone treatment on lymphocyte cell respiration in children with isolated growth hormone deficiency before and after the hormone therapy. A quantitative estimation of the mitochondrial Metabolism, Vol. 28, No. 12 (December), 1979
J. Sherwyn,
S. Amin,
Respiration
and V. T. Maddaiah
effect may be of some help in predicting the response of a given child to growth hormone replacement therapy. MATERIALS
AND
METHODS
Examination and Treatment of Children Out of 28 children that were being followed in the Growth Clinic of the Nassau County Medical Center with the diagnosis of isolated growth hormone deficiency, 12 were included in this study. Parents of these children were informed of the study and gave consent by signing the appropriate forms. Growth hormone treatment was discontinued 4 mo prior to the onset of the study in 8 subjects, and 4 of them had not previously received growth hormone. Growth hormone deficiency was diagnosed by measuring serum hormone levels at different time periods before and after insulin-induced hypoglycemia and stimulation by arginine infusion. Levels of ACTH, TSH, FSH, LH, T,. T,, and CBC were measured in children who showed growth hormone deficiency. Adrenal-pituitary axis was checked by measuring 24-hr urinary levels of 17-ketosteroids and 17ketogenic steroids before and after stimulation by metapyrone. All biochemical analyses were done at the Special Chemistry Laboratories at the Nassau County Medical Center by standard methods. All the children included in the study had normal skull x-ray and girls had normal patterns of buccal smear. X-ray of the left hand and wrist was obtained to evaluate the bone age. The children were put on hormone therapy with 2 IU of hGH (National Pituitary Agency HG-76) administered subcutaneously 3 times a wk and were on no other medication. The children were seen after 8 wk and again after 6 mo. All children were without breakfast the morning of the visit, had gone 24 hr since the last hormone injection, and were examined prior to blood drawing to detect the presence of any infection. Those with infection were excluded from the study. Height was carefully measured against a fixed wall-scale and blood was drawn by the same physician at 9 a.m. at each visit, minimizing diurnal variations.
Isolation of Lymphocytes The method is essentially that developed by Boyurn.” All flasks and syringes were rinsed with 10% EDTA. Approximately 40 ml of venous blood was drawn into a syringe rinsed
From the Department of Pediatrics, Nassau County Medical Center, State University of New York, Stony Brook Health Sciences Center. East Meadow, N. Y. Received for publication March I, 1979. Address reprint requests to V. T. Maddaiah. Ph.D., Department of Pediatrics, Nassau County Medical Center. East Meadow, N. Y. 11554. 0 I979 by Grune & Stratton. Inc. 0026-0495/79/2812-0009$01.00/0 1239
1240
KUNA ET AL.
with 10% EDTA and transferred into the flask containing 1 ml of 10% EDTA. ADP (4 mg/lO ml blood) was added and the samples were incubated at 37OC for 10 min in a rocking incubator chamber to facilitate aggregation of platelets. The blood was layered on top of a chilled mixture of 6% dextran T-500 (Pharmacia) and 39.8% sodium metrizoate solution (2~1) and allowed to stand at O°C. Separation of white cells was complete in about 90 min and all other procedures were performed at O°C. The top brownish layer containing white cells and plasma was gently pipetted out into polyethylene tubes and centrifuged at 4500 g for 10 min. The pellet at the bottom containing white cells was then resuspended by gently blowing air with a bulb-pipette and was subsequently washed three times with the modified Krebs-Ringer solution that was prepared by mixing 0.154-M solutions of NaCl, KCI, MgSO, . 7Hr0, and KHrPO, in the ratio of 9:4: 1: 1 and adjusting the pH to 7.4 with IO M NaOH. The washing procedure removed serum and erythrocytes. Any red button containing hemoglobin and RBC was carefully removed. The final white cell pellet was resuspended in the isolation medium for further use. The cells were examined microscopically using Wright’s stain, and found to be 100% lymphocytes (Fig. 1). Respiration of lymphocytes (l-2 million cells) was monitored polarographically at 30°C with a Clarke-type oxygen electrode (Yellow Springs Instrument, Yellow Springs, Ohio) fitted with a strip chart recorder (Texas Instruments,
Fig. 1.
Houston, Tex.). Incubations were carried out in a jacketed glass chamber containing the respiration medium (isolation medium containing glucose at a final concentration of 0.05%). The whole procedure was terminated by the addition of 1% potassium cyanide solution. The upper limit for 100% oxygen consumption was obtained by adding sodium dithionite powder to the respiration medium. Protein was estimated by the method described by Lowry et a1.l6 using bovine serum albumin as the standard. DNA was estimated by the mithramycin method of Hill and Whately.” Oxygen consumption was expressed as natoms and patoms of oxygen per mg protein and per rg DNA per hour, respectively. All biochemicals were from Sigma Chemical, St. Louis, MO.
RESULTS
Table 1 shows skeletal and chronologic ages of children that were included in the study. The male-female ratio was 8:4. All the children had short stature ranging from -2 to -4 SD below the mean and all had a retarded bone age and isolated growth hormone deficiency. The total white blood counts before and after 6 mo of growth hormone therapy in these children were 6778 and 7142, respectively. Differential counts
A photomicrograph of a smear of a typical preparation of lymphocytes (Wright’s stain, x 400).
GROWTH
HORMONE
ON LYMPHOCYTE
1241
RESPIRATION
Table 1. Growth Characteristics of Subjects Height Increase Height
Subwt
Age (vr)
During
Before
6 mo
Bone Age
Therapy
Therapy
Irl
(inches)
(inches)
sex
1
7
F
4
40%
2%
2
7
M
3
40%
1%
3
12
M
9
50%
1%
4
6
M
4
37%
2%
c
5
3
M
1
29%
1%
al
6
8
M
7
46%
z
2
14
7
M
12
53%
1%
P 0
8
11
F
9
48
1%
E
9
5
F
3
38%
1%
g
10
9
F
7
1%
11
11
M
10
44 42%
12
9
M
6
45%
1%
1
-100’ 1.0
00
&Its Fig. 2. Polarographic tracings of oxygen consumption by isolated lymphocytes (1.8 x 10’ cells1 in a medium (pH 7.41 containing 0.0824 M NaCI, 0.0410 M KCI. 0.0103 M MgSO, 0.0103 M KH,PO. and 0.0028 M glucose. Cells end KCN (final concentration of 0.5 mM were added as indicated.
Inches
30
i6monthsl
granulocytes, monocytes, and lymphocytes (T and B). Some of these factors may have contributed to the large variation in respiration found among the children before and after the therapy. The range of respiratory rates were 14 l-504 natoms of O,/mg protein/hr or 27-l 03 patoms of O,/pg DNA/hr before and after the hormone therapy, respectively. Since the rates varied widely, we chose to relate the percent
2
0
E
+400
-I
5
2
=
+300-
k
atoms
KCN
I”
A plot of percent change in respiration Fig. 3. expressed as natoms of oxygenlhrlmg protein (respiration after growth hormone treatment/respiration before growth hormone treatment x 100). The line is drawn for the best-fitting linear regression equation with the value of correlation coefficient (r) as shown.
.z 2 0z 5 +100P
of oxygen
20
Growth
s ‘$
I
----t t
I
3 75 minutes
I 0.36~
504
1%
were also not significantly different before and after growth hormone treatment (lymphocytes 43% and 45%, granulocytes 57% and 55%). Figure 2 shows a typical oxygen consumption curve. Oxygen consumption was increased after the addition of cells obtained from a child prior to growth hormone therapy. The increase was completely inhibited by KCN, suggesting that the increased consumption was due to mitochondrial respiration. The rate of oxygen consumption was calculated from the slopes.‘* Figure 3 shows a plot of percent change in the rate of respiration/mg protein/hr against increase in height during a period of 6 mo of growth hormone therapy. Lymphocyte respiration may be influenced by several factors, such as isolation techniques, presence of infection, phagocytosis, level of maturation, and relative composition of different types of white cells like
T
-
I 0 E z t a
+200-
. .
o-
-100
0.0
I 1.0
I
2.0
1
3.0
Growthin Inches (6 monthsI Fig. 4. A plot of percent change in respiration expressed as patoms of oxygenlhrlpg DNA (respiration after growth hormone treatment/respiration before growth hormone treatment x 100). The line is drawn for the best-fitting linear regression equation with the value of correlation coefficient (r) as shown
KUNA ET AL.
1242
change in the rate of respiration (respiration after GH treatment/rate of respiration before GH treatment x 100) to growth (increase in height). Correlation coefficient for a linear association between the two parameters (Fig. 2) was 0.667. A plot (not shown) of percent change in respiration against height gain after 8 wk of therapy gave a similar correlation. Figure 4 shows a plot of percent change in respirationlpg DNA/hr against increase in height. A value of 0.756 was obtained for the correlation coefficient for a linear association. Since contamination by DNA of cells other than white cells in the preparation is smaller than contamination by proteins, the values for slope and correlation coefficient are slightly larger for DNA data than for those based on protein. Also, percent change in respiration on the DNA basis was larger than that on a protein basis. DISCUSSION
Although circulating white cells constitute readily available nucleated cells for biochemical studies, they represent a heterogenous group of cells with different structure, function, lifespan, and source of origin.” Different cell types may have different complements of mitochondria. In whole blood, white cells as well as platelets contribute to oxygen consumption. The endogenous respiration of a white cell is approximately 45 times that of an individual platelet. The normal ratio of platelets to white cell is approximately 50: 1. As a result, the amount of oxygen consumed by platelets is equal to white blood cells in a unit volume of blood. Drugs such as thiouracil and chloramphenicol decrease white cell respiration whereas vitamin C increases it. In spite of these and other factors that could affect the rate of oxygen consumption, it is noteworthy that a fairly linear association was
observed between percent change in respiration of lymphocytes and linear growth during growth hormone therapy. Thyroxine is one of the hormones that has been found to influence the metabolic activity of white cells. Hypothyroidism decreased oxygen consumption and the rate of deiodination of thyroxine by white cells, but hyperthyroidism increased both activities.” Farid et al.13 have demonstrated by the qualitative nitroblue-tetrazolium test that polymorphonuclear leukocytes of hypothyroid patients reduced the dye less well when compared to euthyroids. The activity was restored following thyroxine treatment of hypothyroid patients. The present results suggest that growth hormone may also play a role in the regulation of respiratory activity of lymphocytes. The association of increase in respiratory activity of white cells with the increase in height of growth-hormone-deficient children indicates that the mitochondrial effect may be related to the growth-promoting property of the hormone. As discussed elsewhereI with regard to liver mitochondrial activity of experimental animals, growth hormone may influence lymphocyte mitochondrial processes such as protein synthesis, turnover, and component activities of electron transport. Whether these effects are also mediated by somatomedin is not presently known. Since percent change in respiration of lymphocytes measured only 8 wk after growth hormone therapy was also related to height gain, it may become possible to use the early respiratory increment to predict the further efficacy of therapy. ACKNOWLEDGMENT The authors thank the National Pituitary Agency (NIH), for providing human growth hormone, and Pfizer, Inc., Conn., for providing mithramycin.
REFERENCES 1. Maddaiah
VT, Rezvani 1, Chen SY. et al: Distribution of ‘H-acetyl hGH in subcellular fractions of rat liver. Biochem Med 4:492, 1970 2. Groves WE, Houts GE, Bayse GS: Subcellular distribution of ‘2sI-labeled bovine growth hormone in rat liver and kidney. Biochem Biophys Acta 264:472, 1972 3. Maddaiah VT, Sharma RK, Balachandar V, et al: Effect of GH on mitochondrial protein synthesis. J Biol Chem 248:4263,1973 4. Maddaiah VT, Collipp PJ, Lin JH, et al: Growth hormone and liver mitochondria effect on morphology and protein turnover. Biochem Med 16:47, 1976
5. Maddaiah VT, Weston CL, Chen SY, et al: GH and liver mitochondria, effects on cytochromes and some enzymes. Arch Biochem Biophys 173:225, 1976 6. Gupta K, Katkocin DM, Kostyrka ML, et al: Effect of growth hormone on rat liver mitochondria. Fed Proc 36:323, 1977 7. Jemelin M, Frei J: Leukocyte energy metabolism. III anerobic and aerobic ATP production and reiated enzymes. Enzymol Biol Clin 2:289, 1970 8. Frei J, Jemelin M, Aleyassine H: Le metabolisme energetique du leucocyte. Ann Biochem Exp Med 295, 1969
GROWTH
HORMONE
ON LYMPHOCYTE
1243
RESPIRATION
9. McKinney GR, Martin SP, Rindles RW, et al: Respiratory and glycolytic activities of human leukocytes in vitro. J Appl Physiol 5:335, 1953 10. Beck WS. Valentine WN: The carbohydrate metabolism of leukocytes: A review. Cancer Res 13:309, 1953 1 I. Evans AE, Getz GS: Mitochondrial electron transport enzymes in normal leukemic leukocytes. Blood 3 I :7 10, 1968 12. Nessi P, Billesbolle S, Fornerod M, et al: Leukocyte energy metabolism VII. Respiratory chain enzymes, oxygen consumption and oxidative phosphorylation of mitochondria isolated from leukocytes. Enzyme 22:I 83, 1977 13. Farid NR. Au B, Woodford G, et al: Polymorphonuclear leukocyte function in hypothyroidism. Hormone Res 7~247, 1976 14. Maddaiah VT, Collipp PJ: Hormonal regulation of mitochondrial growth, structure and function, in Talwar GP (ed): Regulation of Growth and Differentiated Function in Eukaryote Cells. New York, Raven, 1975, pp 453-459
IS. Boyum A: Separation of leukocytes from blood and bone marrow. Stand J Chn Lab Invest 21 (Suppl97):77-89, 1968 16. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265, 195 1 17. Hill BT, Whately S: A simple rapid DNA. FEBS Lett 56:20, 1975
microassay
for
18. Estabrook RW: Mitochondrial respiratory control and the polarographic measurement of ADP: Ratios. in Estabrook RW, Pullman ME (eds): Methods in Enzymology, vol 10. New York, Academic, 1967, pp 41-47 19. Cline MJ: Metabolism Physiol Rev 45:675, 1965
of the circulating
leukocyte.
20. Kurland GS. Krotkov MV. Freedberg AS: Oxygen consumption and thyroxine deiodination by human leukocytes. J Clin Endocrinol 20:35, 1960