Hypertonic glycerol induces a respiratory burst in leukocytes

Biochimica et BiophysicaActa 882 (1986) 63-70 Elsevier

63

BBA22334

Hypertonic glycerol induces a respiratory burst in leukocytes Mizuho Kaneda and Katsuko Kakinuma The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113 (Japan) (Received September 13th, 1985) (Revised manuscript received March 4th, 1986)

Key words: Respiratory burst; Glycerol; 0 2 ; Lysosomal enzyme; Lactate dehydrogenase; (Leukocyte)

Exposure to hypertonic glycerol induced cyanide-insensitive oxygen consumption and formation of superoxide anion (O2-) in leukocytes such as porcine blood polymorphonuclear leukocytes, guinea pig peritoneal ieukocytes and guinea pig alveolar macrophages. Generation of 02- occurred after a short lag time, remained maximal for a certain time and then stopped. Its termination was not due to cell damage, since cells exposed to glycerol did not release cytosolic enzymes such as lactate dehydrogenase and exhibited a subsequent respiratory burst upon addition of other stimulators such as myristic acid and phorbol myristate acetate. The period of 0 2 generation increased linearly as a function of the glycerol concentration; cells exposed to 20% ( v / v ) glycerol produced 02- for 10 rain. The maximal velocity of 02- generation also increased with the concentration of glycerol, reaching a plateau at 10% glycerol. Membrane vesicles isolated from the cells exposed to 20% glycerol showed high activity of NADPH-dependent O 2- generation as compared to those of unexposed cells. Activation of leukocytes by glycerol was not accompanied by degranulation, unlike stimulation by phagocytosis. A marked change in shape of the cell membrane of glycerol-treated cells was observed by light and scanning electron microscopies.

Introduction Phagocytes such as polymorphonuclear leukocytes, monocytes and macrophages consume oxygen [1,2] for the generation of superoxide anion ( 0 2 ) [3,4] when their plasma membrane is perturbed by phagocytosis or by various kinds of soluble stimulators such as digitonin [5], saponin [6], phorbol myristate acetate [7] or myristic acid [8]. The phenomenon is called 'the respiratory burst'. An NADPH oxidase located in the plasma membrane mediates this oxygen metabolism [9,~0]. Most perturbations of the cell membrane that cause the respiratory burst result in morphological

Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

changes [5] and degranulation of the cell [4]. The respiratory burst can be induced not only by chemical stimulators, but also by a change in osmolarity with 0,09% NaCI solution [111 or with modified Krebs-Ringer phosphate buffer in which Na ÷ is replaced by K ÷ [12]. These results suggest that the plasma membrane-bound N A D P H oxidase is converted from a resting to an active form by a change in osmolarity or an ion exchange through the plasma membrane. In the present work, we report that leukocytes show the respiratory burst when exposed to hypertonic glycerol solution with an iso-osmolar salt composition. Treatment of the leukocytes with this hypertonic glycerol solution did not induce release of lysosomal enzymes, which is in contrast to their activation by chemical stimulators or hypotonic solution. A marked morphological change

0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

64 was observed in the cell surface of the glyceroltreated leukocytes. Materials and Methods

Materials Non-fluorescent glycerol, ethylene glycol, sucrose, polyvinylpyrrolidone K-90, sodium azide, myristic acid (sodium salt) and glutaraldehyde were purchased from Nakarai Chemicals (Kyoto, Japan)• Ferricytochrome c (type VI) and superoxide dismutase (type I) were from Sigma (St. louis, MO, U.S.A.). EDTA was obtained from Dojin Chemicals (Kumamoto, Japan). Radioactive vitamin B-12 ([57Co]cyanocobalamin, 10 #Ci/0.7 /~g) was obtained from Amersham International,, U.K. Other reagents were of analytical grade. Cell preparation Porcine blood polymorphonuclear leukocytes were prepared as described by Wakeyama et al. [13]. Peritoneal leukocytes of guinea pig were prepared from peritoneal exudates obtained 16 h after injection of 5% caseinate-saline [14]. The isolated porcine and guinea pig cells contained 95% polymorphonuclear leukocytes. Preparation of guinea pig alveolar macrophages (more than 90% alveolar macrophages) were isolated from iavage fluid as described previously [15]. Human blood leukocytes were separated from heparinized peripheral blood by dextran sedimentation as reported previously [16]. All prepared cells were suspended in Ca2+-free Krebs-Ringer phosphate buffer (122 mM NaCI/5 mM KC1/1.2 mM MgCI2/17 mM sodium phosphate buffer, pH 7.4) at a concentration of (3-4). 108 cells/ml and placed on ice until use. Metabolic studies Oxygen consumption was measured with a Clark-type oxygen electrode at 37°C. Aliquots of thick cell suspension were mixed with various concentrations of glycerol and 5 mM glucose in Ca z +-free Krebs-Ringer phosphate buffer (pH 7.4) at a concentration of 6 • 106 cells/ml. The rate of 0 2-dependent cytochrome c reduction was measured at 37°C with continuous stirring using a cell mixer [17] by recording the absorption at 550-540 nm with a Hitachi model 556

dual-wavelength spectrophotometer. The reaction mixture contained various concentrations of glycerol, 5 mM glucose, 3 v g / m l catalase and 25 #M ferricytochrome c in CaZ+-free Krebs-Ringer phosphate buffer. The reaction was started by addition of an aliquot of thick cell suspension at a final concentration of 6 • 106 cells/ml. The Oz-dependent reduction of cytochrome c was examined by the addition of superoxide dismutase (20 units/ml). Glycerol was replaced by other hypertonic media such as sodium chloride, ethylene glycol and sucrose in order to examine the hypertonic effect on the 0 2-generating system of leukocytes.

Preparation of membrane vesicles Membrane vesicles were prepared as described previously [18], with modifications• Leukocytes (2 • 107cells/ml) were incubated at 37°C in a hypertonic medium containing 5 mM glucose and 20% glycerol in Krebs-Ringer phosphate buffer (pH 7.4) for 5 min. Control cells were incubated in the buffer without glycerol. The cells exposed to 20% glycerol were suspended in 5 vol. buffer, and centrifuged at 150 Xg for 10 min at 4°C. The sedimented cells were resuspended in ice-cold 0.34 M sucrose and frozen rapidly in solid CO zethanol• The cells were thawed and disrupted by sonication at 40 W for 5 s, followed by centrifugation at 450 x g for 20 min. The postnuclear supernatant was sedimented by centrifugation at 100000 × g for 60 min. The final pellets (membrane vesicles) were suspended in ice-cold 0.34 M sucrose containing 10 mM Hepes buffer (pH 7.0) before use. The Oz-generating activity was measured by superoxide dismutase-inhibitable reduction of cytochrome c at 25°C. The assay mixture contained 25 /~M ferricytochrome c, 3 /~g/ml catalase, membrane vesicles (0.2 mg protein/ml) and 0.17 M sucrose in 65 mM sodium phosphate-potassium phosphate buffer (pH 7.0). The reaction was started by addition of 0.1 mM NADPH in the presence or absence of superoxide dismutase (20 units/ml) [18]. Protein was determined by the method of Lowry et al. with bovine serum albumin as a standard [19]. Enzyme release After incubation of cells with 10-20% (v/v)

65

glycerol at 37°C for 5 min, the cell suspension was cooled in ice-water and centrifuged in an Eppendorf centrifuge 5412 for 10 s. The resulting supernatant was used for assays of extracellular release of azurophilic and specific granule enzymes. Cells incubated at 0°C without glycerol were disrupted by sonication at 0°C for 5 s and solubilized in 0.02% Triton X-100. Enzyme release was expressed as percentage of the total enzyme activity of the solubilized sample. Myeloperoxidase was determined spectrophotometrically by the guaiacol method [20] in the presence of 0.02% Cetavlon [21]. Acid phosphatase and alkaline phosphatase were measured by incubation with p-nitrophenylphosphate as a substrate at pH 5.6 and pH 10.5, respectively [22,23]. Vitamin B-12-binding protein was measured as described previously [24]. Lactate dehydrogenase was measured as the cytoplasmic enzyme, as described previously [25].

Morphological studies Scanning electron microscopy. After incubation with various concentrations of glycerol, the cell suspension was mixed with an equal volume of ice-cold buffer containing 5% glutaraldehyde and the same concentration of glycerol as that for incubation, and then kept at 0°C for 1 h. The samples were then washed with the same glycerol mixture without glutaraldehyde, and allowed to settle onto polylysine-coated cover slips. They were dehydrated in a graded ethanol series, transferred to isoamylacetate, and dried with a critical-point dryer (Hitachi HCP-1). Then they were coated with gold and observed with a scanning electron microscope (Akashi ISI-DS130). Light microscopy. Samples of cell suspension were transferred to glass slides at various times during measurement of 02- generation. Results

Respiratory burst Fig. l a - c shows traces of 02 consumption by porcine blood leukocytes exposed to isotonic buffer, 10%, and 20% glycerol-containing buffer, respectively. 02 consumption started after a short lag time, reached a maximum and, after a few minutes, gradually stopped (Fig. lb and c). When cells were exposed to the buffer in the absence of

cells

a.

b.

-

my

gly

10% gly ~

.

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c. 20% gly

0 -6 E ¢-

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5 min

Fig. 1. Time-courseof 02 consumptionby pig blood ieukocytes exposed to hypertonic glycerol(gly). Consumption of 02 was measured at 37°C with a Clark-type oxygen electrode. The medium contained 10 or 20% (v/v) glycerol,5 mM glucoseand 2 mM sodium azide in Ca2+-frce Krebs-Ringer phosphate buffer (pH 7.4). The reaction was started by addition of an aliquot of cell suspension at a concentrationof 6.106 cells/ml (the first arrow) to 1.8 rrd of buffer solution without glycerol (a), with 10% glycerol(b), or with 20% glycerol(e). After 10 rain, 5/~10.5% sodium myristate(my) dissolved in 50% ethanol was added to the reaction mixture(the second arrow).

glycerol (Fig. la), no respiratory burst was observed. The cells exposed to glycerol were subsequently able to exhibit another respiratory burst on addition of other stimulators, such as phorbol myristate acetate or sodium myristate [8,18]. When exposed to 10% and 20% glycerol the subsequent 02 uptake induced by the addition of myristate was 80% and 33% of that in the absence of glycerol, respectively. The 02 consumption by glyceroltreated cells was resistant to 2 mM azide, like the

66

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Fig. 2. Time-course of O2- release by porcine blood leukocytes exposed to glycerol (gly) -containing buffer. The reaction was started by addition of 30 p.l of cell suspension (107 cells) to 1.6 ml Ca2+-free Krebs-Ringer phosphate buffer, 3 btg/ml of catalase, 25 #M cytochrome c and various concentrations of glycerol: 10% (a) and 20% (b, c). Superoxide dismutase (SOD) (20 units/ml) was added during cytochrome c reduction by 20% glycerol (c).

respiratory burst induced by phagocytosis. Fig. 2a and b show typical tracings of O 2generation by cells incubated at 37°C in KrebsRinger phosphate buffer containing 10% and 20% glycerol. As shown in Fig. 2c, the reduction of cytochrome c by the glycerol-treated cells was completely inhibited by superoxide dismutase. The cells incubated in the buffer without glycerol showed no production of 02- (data not shown). The maximal rate of 02- production of 4 n m o l / m i n per 107 cells after a lag time was seen with cells exposed to 10% glycerol (Fig. 3). The total amount of 02- generation, calculated from the absorption as shown as A A in Fig. 2, increased linearly with concentrations of glycerol of 5-20% as shown in Fig. 4. The period of 0 2 generation (At in Fig. 2) increased linearly as a function of the glycerol concentration; cells exposed to 20% glycerol produced 02- for 10 min. Both the maximum rate and total amount of 02generation decreased with a glycerol concentration of over 30% (data not shown). The effects of hypertonic glycerol on oxidative metabolism were examined with different kinds of

/ I

10 Glycerol (%)

I

20

Fig. 3. Maximal velocity of 0 2 generation by porcine blood leukocytes exposed to glycerol. The reaction mixture was the same as for Fig. 2. Points and vertical bars are means + S.E. for four different samples.

leukocytes as follows. A similar response to 20% glycerol was observed with respect to the 02generation (nmol/min per 107 cells): guinea pig peritoneal polymorphonuclear leukocytes, 5.2 + 1.1 (mean + S.E., n = 3); guinea pig alveolar macrophages, 4.8 (n = 1); human blood polymorphonuclear leukocytes, 0.59 + 0.03 (mean + S.E., n = 3), respectively. Although human blood leukocytes produced little 02- as compared to the other source of leukocytes, the value was apparently larger than that of control cells ( < 0.02 nmol/min per 107 cells). For the test of reversibility, guinea pig peritoneal and porcine leukocytes were pretreated with 20% glycerol for various time intervals: 0.5, 1.0, 4.0 and 10 min, and diluted with 20 vol. KrebsRinger phosphate buffer, followed by centrifugation at 150 x g for 10 min. The washed cells were exposed again to 20% glycerol for measurement of a subsequent respiratory burst. The cells which had been pretreated with 20% glycerol for 30 s showed the second respiratory burst upon exposure to the hypertonic glycerol, which accounted for 40-60% of the first 02- generation. In contrast, the cells pretreated with 20% glycerol over 1.0 min showed no more response to glycerol. In order to examine whether the membranebound NADPH oxidase is activated by exposure

67

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of cells in hypertonic glycerol, membrane vesicles were isolated from glycerol-treated and untreated cells. Membrane vesicles isolated from porcine leukocytes which had been incubated in 20% glycerol for 5 min showed high NADPH-dependent Of-generating activity (14.2 nmol/min per mg protein) as compared to the low activity of those from untreated cells (0.5 nmol/min per mg protein). Fig. 5 shows results on the release of lysosomal enzymes into the medium on exposure of porcine leukocytes to 0, 10 and 20% glycerol for 5 min. The data are expressed as percentages of the following total activities per 107 cells: myeloperoxidase, 2.2 #mol/min; acid phosphatase, 1.0 /~mol/15 rain; alkaline phosphatase, 3.4/~mol/30 min; vitamin B-12-binding protein bound 30.9 ng vitamin B-12; and lactate dehydrogenase, 0.17 nmol/min. The results show that neither

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Fig. 5. Effect of glycerol on release of granule markers and cytosol marker enzyme into the medium. Porcine blood leukocytes (5.106 cells/ml) were incubated at 37°C for 5 min in 0%, 10% or 20% glycerol in Krebs-Ringer phosphate buffer, and then separated into supernatant and pellet fractions by centrifugation. The activity of the supernatant fraction was expressed by the percentage of total activity. MPO, myeloperoxidase; Acid Pase, acid phosphatase; Alkali Pase, alkaline phosphatase; V. B]2 b.p., vitamin B-12-binding protein; LDH, lactate dehydrogenase.

azurophilic nor specific granule markers were released from glycerol-treated cells even on exposure to 10-20% glycerol for 5 min in comparison to control ceils. In addition, no degranulation occurred on exposure to 20% glycerol, even after a prolonged incubation over 15 min (data not shown). The release of lactate dehydrogenase, a cytoplasmic enzyme, was measured to examine whether specific cytolysis occurred in glyceroltreated cells. Only about 2-3% of the total activity was detected in the medium of ceils exposed to either 10 or 20% glycerol for 5 rain. These results indicate that 20% glycerol did not cause cell lysis. When porcine leukocytes were incubated with 30% glycerol for 15 min, they released some lactate dehydrogenase but did not release lysosomal enzymes (data not shown). We also examined the effect of replacing glycerol by other hypertonic materials such as 10-20% sucrose, ethylene glycol and sodium chloride. As shown in Table I, the respiratory

68

burst was induced by the hypertonic sucrose at a concentration above 15%, but not by hypertonic NaC1 or ethylene glycol. Cells exposed to 20% sucrose showed maximal Of generation for a longer period than with glycerol.

TABLE 1 EFFECTS OF H Y P E R T O N I C M A T E R I A L S ON T H E 0 2 GENERATING SYSTEM OF PORCINE BLOOD LEUKOCYTES The assay method was the same as in Fig. 2, except that glycerol was replaced by other materials. The values obtained from the cells exposed to glycerol were means + S.E. for three or four experiments (the number of experiments is given in parentheses) and the values from the cells exposed to sucrose were averages of two experiments. Hypertonic material

Concentration (%)

Maximal rate of 0 2 generation ( n m o l / m i n per 107 cells)

Glycerol

5 10 20

0.6 + 0.3 (3) 3.9 + 0.9 (4) 3.4 _+0.6 (4)

Sucrose

10 15 20 30

0.2 1.9 6.1 4.1

Ethylene glycol

10 2O

0 0

Sodium chloride

5 10 2O

0 0 0

Shape change We examined the morphology of glyceroltreated cells by light microscopy at intervals during the assay of O 2- generation. Untreated porcine leukocytes were round or slightly ruffled before and after the incubation in the buffer without glycerol, but within a few seconds after exposure to 10-20% glycerol, their shape changed markedly; small blebs appeared on their surface, which became larger during maximal stimulation of O zgeneration, and by the time that O z- generation ceased, no round cells were seen. We examined this dramatic shape change of glycerol-treated leukocytes at higher magnification by scanning electron microscopy (Fig. 6a and b). Untreated cells showed small ruffles and some irregularities (Fig. 6a). After treatment with 10% glycerol for 20 s, the cells showed large thin ruffles or projecting bleb-like structures (Fig. 6b). A similar shape change was observed on treating the cells with 10% glycerol for 5 rain before fixation.

E

Fig. 6. Appearance of cells by scanning electron microscopy. (a) Typical control cell fixed with glutaraldehyde after incubation in Krebs-Ringer phosphate buffer without glycerol for 20 s ( x 10000). (b) Typical cell exposed to 10% glycerol in Krebs-Ringer phosphate buffer for 20 s before fixation ( x 10000). Bars, 1.0 #m.

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Discussion The present study showed that hypertonic glycerol caused a marked change in the plasma membrane leading to activation of the plasma membrane-bound NADPH oxidase responsible for the respiratory burst. By light and scanning electron microscopies, we observed that their cell surface changed during the respiratory burst. These findings suggest that glycerol primarily caused physicochemical osmotic shock via the plasma membrane, resulting in modification of the membrane that activates the NADPH oxidase. The respiratory burst of glycerol-treated cells gradually ceased, the period of activity depending on the concentration of glycerol (Figs. 1 and 2). No cytolysis occurred, even on treatment with high concentrations of glycerol, since release of lactate dehydrogenase was negligible (Fig. 5). In fact, the ceils exposed to glycerol showed no morphological evidence of cell rupture as shown in Fig. 6b. Glycerol-treated ceils in which respiratory burst had ceased could again exhibit a respiratory burst on addition of another stimulator such as myristate (Fig. 1). These results suggest that, after activation by hypertonic glycerol, some feedback reaction terminates the respiratory burst, presumably by a modification of membrane constituent(s). To test for reversibility, the cells exposed to 20% glycerol for 1 min were washed by centrifugation before the subsequent exposure to glycerol. Washing by centrifugation caused marked damage to the glycerol-treated cells, so that the washed cells were hardly activated not only by the subsequent treatment with glycerol but also by either myristate or phorbol myristate acetate. Nevertheless, the cells pretreated with 20% glycerol for 30 s showed 40-60% reversibility. In addition, membrane vesicles isolated from the washed cells which had been pretreated with 20% glycerol for 5 rain showed a markedly elevated NADPH oxidase activity as compared to membranes from control cells. Most stimulators of leukocytes that induce the respiratory burst also cause degranulation. However, sodium fluoride [26] and the fifth component of complement, C5a [27] stimulate the respiratory burst without inducing degranulation, suggesting

that the respiratory burst is not connected with degranulation. The present data showed that the respiratory burst induced by glycerol was also not related to degranulation. As with hypertonic glycerol, hypertonic sucrose also caused a respiratory burst (Table I). Cells exposed to hypertonic sucrose showed a similar change in the cell surface, which formed blebs with time. However, hypertonic NaC1 did not activate the 0 2 generation system, possibly because it is a permeable electrolyte; it induced a large, simultaneous efflux of H20 and influx of Na + and CI-. These drastic changes of the intracellular environment probably affected the viability of the cell. In contrast to NaC1, exposure to hypertonic non-electrolytes such as glycerol and sucrose induced a minimum exchange of ions and intracellular water. Ethylene glycol did not induce a respiratory burst (Table I). In addition, ethylene glycol-treated cells did not show another respiratory burst upon addition of myristate (data not shown). It seems likely that ethylene glycol causes a cellular toxicity. High concentrations of sucrose (0.25-0.34 M) have been used for cell homogenization and isolation of cell organelles. The present results indicate that, if leukocytes are suspended in hypertonic sucrose or glycerol, the cells should be kept at a low temperature, otherwise the 0 2 -generating system can be activated. Glycerol has recently been used in a number of biochemical procedures as, for example, an anti-freeze solvent and a stabilizer of enzyme activities. Therefore, further studies seem to be required on the mechanism of its activation of the O2-generating system of the leukocytes.

Acknowledgement We thank Dr. T. Tachibana, Ultrastructural Research Section, and Dr. K. Sato, Biophylaxis Division of our institute, for their technical assistance with electron microscopy. We thank also Dr. T. Yamaguchi of our laboratory, for the assay of vitamin B-12-binding protein.

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