383
Clinica Chimica Acta, 69 (1976) 383-386 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7618
QUANTITATIVE DETERMINATION IN HEALTHY MEN AND WOMEN
Y. HOUPERT,
P. TARALLO
OF GRANULOCYTIC
AMINO ACIDS
and G. SIEST *
Centre de Mkdecine Pre’ventive, 2, Avenue du Doyen Jacques Parisot, 54500 Vandoeuvreles-Nancy et Laboratoire de Biochimie, EUR des Sciences Pharmaceutiques et Biologiques, Rue Albert Lebrun, 54000 Nancy (France) (Received
October
7,
3, 1975)
Summary The concentration of the amino acids has been determined in granulocytes from healthy men and women by chromatographic analysis on ion-exchanging resins. Except taurine, ornithine and aspartic acid, the concentrations of all the amino acids are greater in the cells isolated from women. The most variable amino acids are alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and histidine. Taurine represents 50% of the pool determined from men, whereas for women it represents only 25%.
Introduction The amino acid levels in plasma [1,2,3] and urine [4] are higher in men than in women, except for glycine. Furthermore, the renal excretion of amino acids is related to the plasmatic concentration of oestrogens [ 51. These steroid hormones interfere with the transfer processes of the amino acids across cell membranes [6,7]. The cellular concentrations of amino acids should thus be different between men and women. We have chosen human granulocytes for our study, because they can be isolated very easily. Moreover, they contain most of the amino acids that are usually found in tissues and physiological fluids [8]. Materials and methods Isolation of the granulocytes The granulocytes were obtained * Correspondence Par&t.
from
venous
to: Dr. G. Siest. Centre de MBdecine 54500 Vandoeuvre-les-Nancy, France.
blood,
Pr6ventive.
collected 2. Avenue
on lithium
du Doyen
Jacques
384
heparinate from twenty to forty years old subjects. These subjects were supposedly healthy persons randomly chosen out of the population of Lorraine who came for a check up in the Centre de Medecine Preventive i Vandoeuvreles-Nancy. The preparation of the granulocyte suspension and the assessment of its integrity has already been described [9]. The cell suspension than obtained contained 85 to 95 percent polymorphonuclear leukocytes. No erythrocytes were observed by microscopic examination and the thrombocytic contamination was never more than one platelet for one leukocyte. This contamination by thrombocytes little modified the quantitative determination of the granulocytic amino acids. Indeed, amino acid analysis in platelets was accomplished and showed that the final contamination represented less than ten percent of the quantitative values of the granulocytic amino acids. Amino acid extraction The cell pellet was resuspended in 1 ml of distilled water containing 250 nmol of norleucine which acted as an extraction control and internal standard for the chromatographic analysis of the amino acids. The cells were lysed by three cycles of freezing and thawing. The proteins were denatured by adding 30 mg of 5’-sulfosalicylic acid. The pH of the extract was then adjusted to 1.5. Amino acid analysis The chromatographic amino acid analysis has already been detailed [lo]. We used a Technicon apparatus with a 75 cm glass column of 0.6 cm inner diameter. The resin was a chromobead C, type, diameter 8 pm. The elution was accomplished with a continuous gradient from pH 2.75 to 6.5. The lithium concentrations varied from 0.2 to 1.2 N. The elution temperature is maintained at 40°C during the first 150 min and then fixed at 60°C. Readings were made at 570 nm. The whole analysis lasted 8 h. Results The concentrations of the different amino acids determined from the leukocytes of healthy men and women are listed in Table I. For the women, the stage of the menstrual cycle was not taken into account, and none of them took oral contraceptives. The amino acid pool of women is significantly larger than that of men. The concentrations of alanine, vahne, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and histidine are two to three times higher for women than for men. The small variations of the aspartic acid concentration could result from the chromatographic method used. This procedure does not permit the separation of this compound from the reduced form of glutathione, which was found in very high concentration in the granulocytes [8]. Citrulline was always detected in granulocytes isolated from women, whereas it was found only three times in men. Arginine is present in small quantities in both men and women and its low concentration is due to the presence of an arginase in human leukocytes [ 111. Taurine and ornithine are the only two amino acids, the concentrations of
385
TABLE
I
srmw
OF GRANULOCYTIC
AMINO
Cell isolation and chromatographic
ACIDS
DETERMINED
FOR MEN AND WOMEN
analysis of amino acids were performed as described in Materials and
Methods. Amino acids
Tawine Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine AIanIme CitruIIIne VaIine Methionine Isoleucine Leucine Tyrosine PhenylaIanine Omithine Lysine Histidine A&nine Z nmoI/lO6 ceIIs
Men
t
women
n
Mean (nmol/ 106 ceils.)
o
n
Mean (nmol/ 106 cells)
o
22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22. 10 22
12.38 1.28 0.88 1.19 1.41 1.15 1.14 1.66 Traces 0.70 0.30 0.37 1.07 0.32 0.40 0.35 1.63 0.23 Traces
4.60 0.36 0.41 0.45 0.51 0.31 0.41 0.51 -
12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12
9.06
1.80 0.33 0.53 0.82 0.73 0.48 0.76 0.89 0.29 0.75 0.37 0.64 1.43 0.41 0.52 0.12 0.80 0.29 -
26.48
0.33 0.13 0.19 0.36 0.13 0.16 0.14 0.74 0.11 -
1.40 1.48 1.90 2.40 1.44 2.04 4.13 0.88 1.71 0.85 1.19 3.59 0.91 1.14 0.27 3.53 0.60 Traces
0.02 N.S. 0.001 0.005
Clinica Chimica Acta, 69 (1976) 383-386 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7618
QUANTITATIVE DETERMINATION IN HEALTHY MEN AND WOMEN
Y. HOUPERT,
P. TARALLO
OF GRANULOCYTIC
AMINO ACIDS
and G. SIEST *
Centre de Mkdecine Pre’ventive, 2, Avenue du Doyen Jacques Parisot, 54500 Vandoeuvreles-Nancy et Laboratoire de Biochimie, EUR des Sciences Pharmaceutiques et Biologiques, Rue Albert Lebrun, 54000 Nancy (France) (Received
October
7,
3, 1975)
Summary The concentration of the amino acids has been determined in granulocytes from healthy men and women by chromatographic analysis on ion-exchanging resins. Except taurine, ornithine and aspartic acid, the concentrations of all the amino acids are greater in the cells isolated from women. The most variable amino acids are alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and histidine. Taurine represents 50% of the pool determined from men, whereas for women it represents only 25%.
Introduction The amino acid levels in plasma [1,2,3] and urine [4] are higher in men than in women, except for glycine. Furthermore, the renal excretion of amino acids is related to the plasmatic concentration of oestrogens [ 51. These steroid hormones interfere with the transfer processes of the amino acids across cell membranes [6,7]. The cellular concentrations of amino acids should thus be different between men and women. We have chosen human granulocytes for our study, because they can be isolated very easily. Moreover, they contain most of the amino acids that are usually found in tissues and physiological fluids [8]. Materials and methods Isolation of the granulocytes The granulocytes were obtained * Correspondence Par&t.
from
venous
to: Dr. G. Siest. Centre de MBdecine 54500 Vandoeuvre-les-Nancy, France.
blood,
Pr6ventive.
collected 2. Avenue
on lithium
du Doyen
Jacques
384
heparinate from twenty to forty years old subjects. These subjects were supposedly healthy persons randomly chosen out of the population of Lorraine who came for a check up in the Centre de Medecine Preventive i Vandoeuvreles-Nancy. The preparation of the granulocyte suspension and the assessment of its integrity has already been described [9]. The cell suspension than obtained contained 85 to 95 percent polymorphonuclear leukocytes. No erythrocytes were observed by microscopic examination and the thrombocytic contamination was never more than one platelet for one leukocyte. This contamination by thrombocytes little modified the quantitative determination of the granulocytic amino acids. Indeed, amino acid analysis in platelets was accomplished and showed that the final contamination represented less than ten percent of the quantitative values of the granulocytic amino acids. Amino acid extraction The cell pellet was resuspended in 1 ml of distilled water containing 250 nmol of norleucine which acted as an extraction control and internal standard for the chromatographic analysis of the amino acids. The cells were lysed by three cycles of freezing and thawing. The proteins were denatured by adding 30 mg of 5’-sulfosalicylic acid. The pH of the extract was then adjusted to 1.5. Amino acid analysis The chromatographic amino acid analysis has already been detailed [lo]. We used a Technicon apparatus with a 75 cm glass column of 0.6 cm inner diameter. The resin was a chromobead C, type, diameter 8 pm. The elution was accomplished with a continuous gradient from pH 2.75 to 6.5. The lithium concentrations varied from 0.2 to 1.2 N. The elution temperature is maintained at 40°C during the first 150 min and then fixed at 60°C. Readings were made at 570 nm. The whole analysis lasted 8 h. Results The concentrations of the different amino acids determined from the leukocytes of healthy men and women are listed in Table I. For the women, the stage of the menstrual cycle was not taken into account, and none of them took oral contraceptives. The amino acid pool of women is significantly larger than that of men. The concentrations of alanine, vahne, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and histidine are two to three times higher for women than for men. The small variations of the aspartic acid concentration could result from the chromatographic method used. This procedure does not permit the separation of this compound from the reduced form of glutathione, which was found in very high concentration in the granulocytes [8]. Citrulline was always detected in granulocytes isolated from women, whereas it was found only three times in men. Arginine is present in small quantities in both men and women and its low concentration is due to the presence of an arginase in human leukocytes [ 111. Taurine and ornithine are the only two amino acids, the concentrations of
385
TABLE
I
srmw
OF GRANULOCYTIC
AMINO
Cell isolation and chromatographic
ACIDS
DETERMINED
FOR MEN AND WOMEN
analysis of amino acids were performed as described in Materials and
Methods. Amino acids
Tawine Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine AIanIme CitruIIIne VaIine Methionine Isoleucine Leucine Tyrosine PhenylaIanine Omithine Lysine Histidine A&nine Z nmoI/lO6 ceIIs
Men
t
women
n
Mean (nmol/ 106 ceils.)
o
n
Mean (nmol/ 106 cells)
o
22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22. 10 22
12.38 1.28 0.88 1.19 1.41 1.15 1.14 1.66 Traces 0.70 0.30 0.37 1.07 0.32 0.40 0.35 1.63 0.23 Traces
4.60 0.36 0.41 0.45 0.51 0.31 0.41 0.51 -
12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12
9.06
1.80 0.33 0.53 0.82 0.73 0.48 0.76 0.89 0.29 0.75 0.37 0.64 1.43 0.41 0.52 0.12 0.80 0.29 -
26.48
0.33 0.13 0.19 0.36 0.13 0.16 0.14 0.74 0.11 -
1.40 1.48 1.90 2.40 1.44 2.04 4.13 0.88 1.71 0.85 1.19 3.59 0.91 1.14 0.27 3.53 0.60 Traces
0.02 N.S. 0.001 0.005
