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The real and apparent plasma oxalate
305
Clinica Chimico Acta, 101 (1960) 305-311 @ Elsevier/North-Holland Biomedical Press
CCA 1269
THE REAL AND APPARENT PLASMA OXALATE
T. AKCAY a+band G. ALAN ROSE *
St. Peter’s Hospital and Institute of Urology, London
(U.K.)
(Received June 4th, 1979)
A method for the determination of human plasma oxalate concentration by an enzymatic assay procedure is described using deproteinised plasma. The apparent concentration of oxalate in 20 normal subjects was 1.1-16.0 pmol/l (mean 7.78; S.D. 3.96). It was suspected that these results might be too high, due to the possible conversion of glyoxalate to oxalate, and this reaction was clearly demonstrated to occur in whole blood in vitro. Inhibitors (boric acid, DL-fl-phenyllactic acid, and allopurinol) of this oxidation were therefore added to the freshly taken blood samples, prior to assaying by the same technique. The plasma oxalate concentration from normal subjects was then found to be O-5.4 pmol/l (mean 2.26; S.D. 1.67). It is concluded that normal blood spontaneously generates oxalate on standing, and the higher values obtained by other in vitro methods must be fallacious.
Introduction A variety of methods have been used for measurement of oxalate in plasma and these were reviewed in 1977 by Hodgkinson [ 11. Nevertheless, there is still disagreement about the normal range. In vivo human isotopic methods have yielded values of approximately 1 pmol/l in several different laboratories [ 2-41, whereas chemical methods have yielded values at least 10 times higher in various laboratories [ 5-81. In order to try and resolve this discrepancy, we ‘modified the enzymatic method of Hallson and Rose [9] that had been in use in these laboratories for some six years for the measurement of urinary oxalate, so as to make it sufficiently sensitive to detect the much lower concentration * Correspondence to G. Alan Rose, St. Paul’s Hospital, Endell Street, London, WC2. a Present address: Cermhpasa Tip Fakultesi. Biokimya Ktirsirti, Uzman Asistan, Aksaray, Istanbul, Tur key. b In receipt of a grant from the University of Istanbul.
306
of normal plasma. These modifications are reported here. Preliminary results then showed plasma oxalate levels of S-15 I.tmol/l. Since red blood cells can oxidise added glyoxylate to oxalate [lo], it occurred to us that oxalate might be generated spontaneously in blood, perhaps by oxidation of glyoxylate. Glyoxylate added to blood was then shown to be oxidised to oxalate, and on adding inhibitors of glyoxylate oxidation to the syringe used for venepuncture and collection of blood, the plasma samples showed much lower oxalate values. These results are also reported in this paper. Material and methods Plasma oxalate concentrations were determined by the enzymatic method of Hallson and Rose [9], but with the following modifications in order to increase the sensitivity: (1) Citrate was found to cause a significant blank with the enzyme oxalate decarboxylase, as also noted by Sallis [ 111, and was, therefore, replaced in the acid buffer by phthalate (pH 4; 0.1 mol/l). It was shown that phthalate buffer interfered in no way with the enzyme system. (2) The concentration of the alkaline buffer (prepared from sodium carbonate) was reduced to 0.06 mmol/l. This was freshly prepared prior to each assay. (3) The concentration of the working standard oxalic acid solution was 110 pmol/l. (4) The assay was carried out at 22°C rather than 37”C, as the former temperature gave a larger change in pH and was more convenient. (5) In order to reduce the blank further, each sample and blank was incubated for two nights. During the first night no oxalate decarboxylase was present, and the inner tube contained soda lime instead of the weak alkaline buffer. (6) The inhibitor employed to prevent the enzymatic conversion of glyoxylate to oxalate consisted of boric acid 71.9 mmol (4.44 g), allopurinol 1.76 mmol (240 mg), and DL-fl-phenyllactic acid 27.1 mmol (4.5 g) made up to 100 ml with distilled water and adjusted to pH 7.4. (7) Lower blanks were obtained after deproteinisation of plasma. The method used was to mix equal volumes of plasma and 0.05 mol/l hydrochloric acid, and heat at 100°C for 5 min and remove the precipitate by centrifuging. Assays were performed on the clear supernatant solution. Procedure
Plasma samples were obtained either from normal non-fasting individuals or by pooling samples from patients whose plasma creatinine levels had already been measured for other purposes. In the case of the former, 50 ml of venous blood was withdrawn into a heparinised syringe which contained 5 ml of the combined inhibitor solution. The syringe was gently rotated while the blood was being withdrawn, so as to obtain as rapid mixing as possible. The blood was then immediately centrifuged at 4”C, after which equal volumes of 0.05 mol/l HCl and plasma were heated in a water bath at 100°C for 5 min. The deproteinised plasma (DPP) was adjusted to pH 3.8 by the addition of dilute hydro-
307
chloric acid. Dissolved CO, was removed from the DPP firstly on the vacuum pump and then by passing through it a rapid stream of oxygen. The CO1-free DPP was divided into six equal aliquots. Three were used for the assay, of plasma oxalate and the other three were blank controls. Each aliquot was placed in an individual conical flask [9] containing inner tubes filled with soda lime, flushed with oxygen, stoppered and incubated at 22°C overnight. The next day, the soda lime tubes were removed and replaced by similar tubes containing 2 ml of alkaline buffer. 1 ml of CO*-free enzyme solution was added to each flask used for the oxalate assay. Each of the other flasks received CO*-free phthalate buffer. Thereafter the procedure was substantially as previously described [9], except that to read final pH it was necessary to transfer the alkaline buffer anaerobically to a flow-through electrode (Pye-unicam, Cat. No. 402-611). A calibration curve was obtained by using 0.2 and 0.5 ml of working standard solution. The sensitivity of the method is 11 nmol/l (1 lug) of oxalatic acid. Results The results are shown on Tables I-IV. A. Without inhibitors of glyoxylate oxidation. The mean of the plasma oxalate values obtained without deproteinisation on 6 batches of pooled plasma from individuals with normal plasma creatinine levels and without known disturbance, of oxalate metabolism was 11.05 pmol/l (S.D. 2.84): As seen in Table I, in 2 further pools of plasma from patients with chronic renal failure, the plasma o&late values obtained in the same way rose progressively with rising creatinine levels. When samples of plasma were studied with deproteinisation, the mean of 6 pools with normal creatinine levels was 11.48 pmol/l (S.D. 1.79). Nineteen other individual samples of plasma from normal volunteers were also studied after deproteinisation and the mean of these oxalate levels was 7.55 pmol/l (SD. 4.13). Again, the observed oxalate level rose with renal failure. TABLE PLASMA
I OXALATE
VALUES
OBTAINED Plasma wmwu
A.
Undeproteinised
12.8 8.2 8.6
B. Deproteinised
oxa1ate
FROM
POOLED
PLASMA
Plasma (mmol/l) 0.09 0.05 0.09
15.5
0.09
9.4
0.08
11.8
0.07
46
0.90
33.3
1.30
23.1
0.3
30.0
0.8
42.2
1.2
creatinine
WITHOUT
ANY
INHIBITORS
308
TABLE PLASMA
II OXALATE
VALUES Without
Pool
A
Pool
B
14.4
Pool
c
11.1
Pool
D
12.2
Pool
E
9.1
(pmol/l)
FROM
NORMAL With
inhibitors
SUBJECTS
USING
DEPROTEINISATION
inhibitors
11.7
B.C.
6.2
2.7
GUY
5.4
5.4
NU.
1.1
1.1
O.R.
15.2
4.8
G.L.
16.0
J.K.
10.0
O.R.
9.4
C.S.
10.0
Nu.T.
7.6
B.H.
8.5
P.Ha.
3.4
P.He.
1.9
E.W. A.A.
3.81
1.8
B. With inhibitors of glyoxylate oxidation. Eleven plasma samples were studied in normal individuals using deproteinisation, and the mean of the values for oxalate was 2.26 E.tmol/l(S.D. 1.67). C. Recovery of added oxalate. Four recovery studies were performed, in which plasma oxalate was determined with and without addition of known quntities of oxalate. One study was on pooled plasma and did not use inhibitors. The other studies were on individual samples from normal volunteers and did use inhibitors. As shown in Table IV, the recovery appeared to be slightly
TABLE EFFECT
III OF
ADDED
GLYOXYLATE
Glyoxylate
ON
added
APPARENT
Oxalate
found
PLASMA
OXALATE
WITHOUT
(@mol/l)
Conversion oxalate
(ma Without A.
Glyoxylate
INHIBITORS
added
to separated
glyoxylate
With
glyoxylate
(96)
plasma
NU
1.36
*
3.8
4.5
0.16
LW
1.65
*
2.1
4.6
0.34
C.S.
I.10
*
4.4
5.0
0.08
100
0.38
B. Glyoxylate
added
to whole
blood
P.H.
(59.7)
*
7.3
C.S.
(11.9)
*
10.6
92.8
1.72
T.O.
(2.2)
*
11.3
67.2
6.3
O.R.
(2.2)
*
9.1
65.7
6.4
S.S.
(2.4)
*
4.7
40.0
3.6
* mg.
to
309 TABLE IV RECOVERY
OF OXALATE
FROM PLASMA Oxalate (/?mml/l)
A. Pooled plasma noinhibitor * M.Na ** with inhibitor D. ** with inhibitor P.G. ** with inhibitor
Recovery (96)
Without additive
Cont. added
Total found
10.4 1.5 1.3 0.95
22.3 5.5 5.5 5.5
32.1 6.1 6.6 6.0
102 92 96 90 95
Mean * Oxalate added after deproteinisation. ** Oxalate added before deproteinisation.
lower after deproteinisation,
but this is probably not significantly different. on observed oxahte levels (Table III). In these studies two syringes were used to withdraw a total of 100 ml blood from normal subjects. One syringeful was treated in the usual way, and the other was used for addition of glyoxylate. In three cases the glyoxylate, in the form of a solution of the neutral sodium salt, was added to 25 ml of the fresh plasma immediately after separation from the red cells. After incubation at room temperature for 1 h, the oxalate level was measured in the usual way. As shown in Table III, the conversion of glyoxylate to oxalate by plasma was small and only just detectable. In five cases the glyoxylate was added.to the syringes prior to withdrawal of blood from the normal subjects. This blood was immediately centrifuged at room temperature and the plasma then analysed in the usual way. As shown in Table III, there was a large conversion of glyoxylate to oxalate by whole blood. D. Effect
of added glyoxylate
Discussion Whether or not glyoxylate occurs in plasma is not known for certain. However, it does occur in urine and, therefore, it seems reasonable to suppose it also occurs in plasma. The amount in urine is about 0.02 to 0.06 mmol in 24 h [ 121, about l/5 of the normal urinary glycollate excretion [13], and it may be that the plasma levels of the two acids are similarly related. Since the amount of normal plasma glycollate is about 0.2 mmol/l [13], a suggested figure for the normal plasma glyoxylate is about 0.04 mmol/l, a figure far higher than that for any current estimates of normal plasma oxalate. Hence, if there was any significant tendency for glyoxylate to be oxidised in the blood to oxalate, then measurements of plasma oxalate in vitro would be invalidated and come out ‘much too high. Glyoxylate is oxidised to oxalate by three enzymes, namely xanthine oxidase [ 141, lactic dehydrogenase (LDH) [ 151 and glycollate oxidase [ 161. The first two of these enzymes are known to be present in plasma, and the third may be; hence, there seems to be a prima facie case for supposing that glyoxylate in plasma would interfere with the oxalate assay. In fact, it has been
310
confirmed here, that glyoxylate does not itself interfere with the oxalate assay, when in aqueous solution free from enzymes, as previously reported [9]. On the other hand, when glyoxylate was added to plasma, the apparent oxalate level was slightly increased, and when added to whole blood, the level was enormously increased (Table III). Xanthine oxidase can be inhibited by allopurinol [17], LDH by borate [ 181 and glycollate oxidase by phenyllactate [ 191, and it was, therefore, decided to try the effect of adding a mixture of these three inhibitors to the syringe used for venepuncture. Table II shows that this addition resulted in a considerable fall in the apparent oxalate level, although it had no effect on the assay of oxalate in aqueous solutions. It therefore seems reasonable to suppose that the inhibitors were suppressing formation of oxalate in plasma or blood in vitro and that the probable causeof such additional oxalate was glyoxylate. These results seem to show that no chemical method of measuring plasma oxalate at normal levels in vitro can be accurate, unless a method of blocking further formation of oxalate is used. It is therefore clear that all previous normal figures quoted for in vitro determination of oxalate are too high, and it seems likely that the lower in vivo isotopic determinations are correct. The normal values now reported are the lowest yet obtained by an in vitro method and they are only slightly higher than those found by in vivo isotopic methods [l]. It is possible that even these are too high, as we cannot be certain that inhibition of oxidation of glyoxylate was always complete. It is not easy to ensure instantaneous mixing of inhibitor and blood in the syringe and there could be other reasons as well. Measurements of oxalate at such low levels as now reported in normal plasma are at the limits of sensitivity of the method and are not as accurate as one would like. It remains to be explained why in chronic renal failure the in vivo and in vitro methods appear to agree [4]. It is known that LDH activity in uraemic serum is reduced by dialysable substances, thought to be urea and oxalate [20], and oxalate itself inhibits the oxidation of glyoxylate to oxalate [lo]. As it seems that both plasma urea and oxalate are greatly increased in chronic renal failure, it may well be that the spontaneous oxidation of glyoxylate to oxalate does not take place in the blood of patients with this condition. Furthermore, since the plasma oxalate is so much higher in chronic renal failure the effect of oxidation of glyoxylate to oxalate would be much less apparent. It should be pointed out that the measurement of plasma oxalate by the method described is highly demanding, tedious, and requires a large volume of blood. It is, therefore, not recommended for routine laboratory use, but does seem to have a place in research projects such as that described here. Acknowledgements We are grateful to Wellcome Laboratories Ltd. for the gift of Allopurinol powder, and to the normal volunteers who donated their blood so freely.
311
References 1
Hodgkinson,
2
Williams,
3
Hodgkinson.
4
Constable, Med.
5
Knowles, Krugers
(1977)
Oxalic
Johnson. A.
Acid
G.A.
and
and Wilkinson,
R.
A.R..
56.
6
A. H.E.,
Joekes.
A.M.,
in Biology
and
Medicine.
Smith,
L.H.
(1971)
(1974)
Clin.
Sci.
Kasidas.
G.P..
Academic
Clin. Molec.
O’Regan,
Press,
Sci.
41.
213-218
Med.
46,
61-73
P.
and
Rose,
G.A.
London
(1979)
Clin.
Sci.
Molec.
299-304 C.F.
and
Hodgkinson,
Dagneaux,
A.
P.G.L.C..
(1972)
Klein
Analyst
Elhorst,
97,474481
J.T.
and
Olthuis,
F.M.F.G.
(1976)
Clin.
Chim.
Acta
71.
319-325 7
Hatch,
M.,
8
Nuret,
P. and
Bourk,
9
Hallson,
P.C.
10
Fisher.
V.
11
Sallis.
d.D.,
12
Hockaday,
E. and
Offner. and
Rose.
and Watts,
(1978) G.A.
R.W.E.
Lumley, T.D.R.,
Costello,
M.
M.F.
J. (1977) Clin.
Chim.
Clin.
Chem.
Acta
82.
9-12
Acta
55.
(1974)
Clin.
Chim.
(1968)
Clin.
Sci.
and
Jordan.
Frederick,
E.W.,
J.E.
34,
76-78
29-39
97-110
(1977)
Clayton,
23,
Biochem.
J.E.
and
Med.
Smith,
18.
L.H.
371-377
(1965)
J. Lab.
Clin.
Med.
65,
677-
687 13
Kasidas,
14
Booth,
G.P.
15
Banner,
16
Richardson,
17
Elion,
18
Nachlas,
19
Liao,
20
Wilkinson.
V.H.
and
M.R.
L.L.
and K.E.
B.G.
Rose,
(1938)
Rosalki, and
(1966)
G.A.
Ann.
Margulies,
and
Richardson, Fujimoto.
Clin.
J. 32.
S.B.
Tolbert,
M.M., J.H.,
(1979)
Biochem.
Rheum. S.I.. K.E. Y.,
(1967) N.E.
Chin
Acta
96,
25-36
494-502 Nature
(1961)
Dis.
25,
Goldberg. (1973) Senesky,
213,
J. Biol.
726-727 Chem.
236.
1280-1284
608 J.D. Arch. D. and
and
Seligman,
Biochem. Ludwig,
M.A.
Biophys. G.D.
(1960)
Anal.
Biochem.
1, 317-326
154,68-75
(1970)
J. Lab.
Clin.
Med.
75,
109
Clinica Chimico Acta, 101 (1960) 305-311 @ Elsevier/North-Holland Biomedical Press
CCA 1269
THE REAL AND APPARENT PLASMA OXALATE
T. AKCAY a+band G. ALAN ROSE *
St. Peter’s Hospital and Institute of Urology, London
(U.K.)
(Received June 4th, 1979)
A method for the determination of human plasma oxalate concentration by an enzymatic assay procedure is described using deproteinised plasma. The apparent concentration of oxalate in 20 normal subjects was 1.1-16.0 pmol/l (mean 7.78; S.D. 3.96). It was suspected that these results might be too high, due to the possible conversion of glyoxalate to oxalate, and this reaction was clearly demonstrated to occur in whole blood in vitro. Inhibitors (boric acid, DL-fl-phenyllactic acid, and allopurinol) of this oxidation were therefore added to the freshly taken blood samples, prior to assaying by the same technique. The plasma oxalate concentration from normal subjects was then found to be O-5.4 pmol/l (mean 2.26; S.D. 1.67). It is concluded that normal blood spontaneously generates oxalate on standing, and the higher values obtained by other in vitro methods must be fallacious.
Introduction A variety of methods have been used for measurement of oxalate in plasma and these were reviewed in 1977 by Hodgkinson [ 11. Nevertheless, there is still disagreement about the normal range. In vivo human isotopic methods have yielded values of approximately 1 pmol/l in several different laboratories [ 2-41, whereas chemical methods have yielded values at least 10 times higher in various laboratories [ 5-81. In order to try and resolve this discrepancy, we ‘modified the enzymatic method of Hallson and Rose [9] that had been in use in these laboratories for some six years for the measurement of urinary oxalate, so as to make it sufficiently sensitive to detect the much lower concentration * Correspondence to G. Alan Rose, St. Paul’s Hospital, Endell Street, London, WC2. a Present address: Cermhpasa Tip Fakultesi. Biokimya Ktirsirti, Uzman Asistan, Aksaray, Istanbul, Tur key. b In receipt of a grant from the University of Istanbul.
306
of normal plasma. These modifications are reported here. Preliminary results then showed plasma oxalate levels of S-15 I.tmol/l. Since red blood cells can oxidise added glyoxylate to oxalate [lo], it occurred to us that oxalate might be generated spontaneously in blood, perhaps by oxidation of glyoxylate. Glyoxylate added to blood was then shown to be oxidised to oxalate, and on adding inhibitors of glyoxylate oxidation to the syringe used for venepuncture and collection of blood, the plasma samples showed much lower oxalate values. These results are also reported in this paper. Material and methods Plasma oxalate concentrations were determined by the enzymatic method of Hallson and Rose [9], but with the following modifications in order to increase the sensitivity: (1) Citrate was found to cause a significant blank with the enzyme oxalate decarboxylase, as also noted by Sallis [ 111, and was, therefore, replaced in the acid buffer by phthalate (pH 4; 0.1 mol/l). It was shown that phthalate buffer interfered in no way with the enzyme system. (2) The concentration of the alkaline buffer (prepared from sodium carbonate) was reduced to 0.06 mmol/l. This was freshly prepared prior to each assay. (3) The concentration of the working standard oxalic acid solution was 110 pmol/l. (4) The assay was carried out at 22°C rather than 37”C, as the former temperature gave a larger change in pH and was more convenient. (5) In order to reduce the blank further, each sample and blank was incubated for two nights. During the first night no oxalate decarboxylase was present, and the inner tube contained soda lime instead of the weak alkaline buffer. (6) The inhibitor employed to prevent the enzymatic conversion of glyoxylate to oxalate consisted of boric acid 71.9 mmol (4.44 g), allopurinol 1.76 mmol (240 mg), and DL-fl-phenyllactic acid 27.1 mmol (4.5 g) made up to 100 ml with distilled water and adjusted to pH 7.4. (7) Lower blanks were obtained after deproteinisation of plasma. The method used was to mix equal volumes of plasma and 0.05 mol/l hydrochloric acid, and heat at 100°C for 5 min and remove the precipitate by centrifuging. Assays were performed on the clear supernatant solution. Procedure
Plasma samples were obtained either from normal non-fasting individuals or by pooling samples from patients whose plasma creatinine levels had already been measured for other purposes. In the case of the former, 50 ml of venous blood was withdrawn into a heparinised syringe which contained 5 ml of the combined inhibitor solution. The syringe was gently rotated while the blood was being withdrawn, so as to obtain as rapid mixing as possible. The blood was then immediately centrifuged at 4”C, after which equal volumes of 0.05 mol/l HCl and plasma were heated in a water bath at 100°C for 5 min. The deproteinised plasma (DPP) was adjusted to pH 3.8 by the addition of dilute hydro-
307
chloric acid. Dissolved CO, was removed from the DPP firstly on the vacuum pump and then by passing through it a rapid stream of oxygen. The CO1-free DPP was divided into six equal aliquots. Three were used for the assay, of plasma oxalate and the other three were blank controls. Each aliquot was placed in an individual conical flask [9] containing inner tubes filled with soda lime, flushed with oxygen, stoppered and incubated at 22°C overnight. The next day, the soda lime tubes were removed and replaced by similar tubes containing 2 ml of alkaline buffer. 1 ml of CO*-free enzyme solution was added to each flask used for the oxalate assay. Each of the other flasks received CO*-free phthalate buffer. Thereafter the procedure was substantially as previously described [9], except that to read final pH it was necessary to transfer the alkaline buffer anaerobically to a flow-through electrode (Pye-unicam, Cat. No. 402-611). A calibration curve was obtained by using 0.2 and 0.5 ml of working standard solution. The sensitivity of the method is 11 nmol/l (1 lug) of oxalatic acid. Results The results are shown on Tables I-IV. A. Without inhibitors of glyoxylate oxidation. The mean of the plasma oxalate values obtained without deproteinisation on 6 batches of pooled plasma from individuals with normal plasma creatinine levels and without known disturbance, of oxalate metabolism was 11.05 pmol/l (S.D. 2.84): As seen in Table I, in 2 further pools of plasma from patients with chronic renal failure, the plasma o&late values obtained in the same way rose progressively with rising creatinine levels. When samples of plasma were studied with deproteinisation, the mean of 6 pools with normal creatinine levels was 11.48 pmol/l (S.D. 1.79). Nineteen other individual samples of plasma from normal volunteers were also studied after deproteinisation and the mean of these oxalate levels was 7.55 pmol/l (SD. 4.13). Again, the observed oxalate level rose with renal failure. TABLE PLASMA
I OXALATE
VALUES
OBTAINED Plasma wmwu
A.
Undeproteinised
12.8 8.2 8.6
B. Deproteinised
oxa1ate
FROM
POOLED
PLASMA
Plasma (mmol/l) 0.09 0.05 0.09
15.5
0.09
9.4
0.08
11.8
0.07
46
0.90
33.3
1.30
23.1
0.3
30.0
0.8
42.2
1.2
creatinine
WITHOUT
ANY
INHIBITORS
308
TABLE PLASMA
II OXALATE
VALUES Without
Pool
A
Pool
B
14.4
Pool
c
11.1
Pool
D
12.2
Pool
E
9.1
(pmol/l)
FROM
NORMAL With
inhibitors
SUBJECTS
USING
DEPROTEINISATION
inhibitors
11.7
B.C.
6.2
2.7
GUY
5.4
5.4
NU.
1.1
1.1
O.R.
15.2
4.8
G.L.
16.0
J.K.
10.0
O.R.
9.4
C.S.
10.0
Nu.T.
7.6
B.H.
8.5
P.Ha.
3.4
P.He.
1.9
E.W. A.A.
3.81
1.8
B. With inhibitors of glyoxylate oxidation. Eleven plasma samples were studied in normal individuals using deproteinisation, and the mean of the values for oxalate was 2.26 E.tmol/l(S.D. 1.67). C. Recovery of added oxalate. Four recovery studies were performed, in which plasma oxalate was determined with and without addition of known quntities of oxalate. One study was on pooled plasma and did not use inhibitors. The other studies were on individual samples from normal volunteers and did use inhibitors. As shown in Table IV, the recovery appeared to be slightly
TABLE EFFECT
III OF
ADDED
GLYOXYLATE
Glyoxylate
ON
added
APPARENT
Oxalate
found
PLASMA
OXALATE
WITHOUT
(@mol/l)
Conversion oxalate
(ma Without A.
Glyoxylate
INHIBITORS
added
to separated
glyoxylate
With
glyoxylate
(96)
plasma
NU
1.36
*
3.8
4.5
0.16
LW
1.65
*
2.1
4.6
0.34
C.S.
I.10
*
4.4
5.0
0.08
100
0.38
B. Glyoxylate
added
to whole
blood
P.H.
(59.7)
*
7.3
C.S.
(11.9)
*
10.6
92.8
1.72
T.O.
(2.2)
*
11.3
67.2
6.3
O.R.
(2.2)
*
9.1
65.7
6.4
S.S.
(2.4)
*
4.7
40.0
3.6
* mg.
to
309 TABLE IV RECOVERY
OF OXALATE
FROM PLASMA Oxalate (/?mml/l)
A. Pooled plasma noinhibitor * M.Na ** with inhibitor D. ** with inhibitor P.G. ** with inhibitor
Recovery (96)
Without additive
Cont. added
Total found
10.4 1.5 1.3 0.95
22.3 5.5 5.5 5.5
32.1 6.1 6.6 6.0
102 92 96 90 95
Mean * Oxalate added after deproteinisation. ** Oxalate added before deproteinisation.
lower after deproteinisation,
but this is probably not significantly different. on observed oxahte levels (Table III). In these studies two syringes were used to withdraw a total of 100 ml blood from normal subjects. One syringeful was treated in the usual way, and the other was used for addition of glyoxylate. In three cases the glyoxylate, in the form of a solution of the neutral sodium salt, was added to 25 ml of the fresh plasma immediately after separation from the red cells. After incubation at room temperature for 1 h, the oxalate level was measured in the usual way. As shown in Table III, the conversion of glyoxylate to oxalate by plasma was small and only just detectable. In five cases the glyoxylate was added.to the syringes prior to withdrawal of blood from the normal subjects. This blood was immediately centrifuged at room temperature and the plasma then analysed in the usual way. As shown in Table III, there was a large conversion of glyoxylate to oxalate by whole blood. D. Effect
of added glyoxylate
Discussion Whether or not glyoxylate occurs in plasma is not known for certain. However, it does occur in urine and, therefore, it seems reasonable to suppose it also occurs in plasma. The amount in urine is about 0.02 to 0.06 mmol in 24 h [ 121, about l/5 of the normal urinary glycollate excretion [13], and it may be that the plasma levels of the two acids are similarly related. Since the amount of normal plasma glycollate is about 0.2 mmol/l [13], a suggested figure for the normal plasma glyoxylate is about 0.04 mmol/l, a figure far higher than that for any current estimates of normal plasma oxalate. Hence, if there was any significant tendency for glyoxylate to be oxidised in the blood to oxalate, then measurements of plasma oxalate in vitro would be invalidated and come out ‘much too high. Glyoxylate is oxidised to oxalate by three enzymes, namely xanthine oxidase [ 141, lactic dehydrogenase (LDH) [ 151 and glycollate oxidase [ 161. The first two of these enzymes are known to be present in plasma, and the third may be; hence, there seems to be a prima facie case for supposing that glyoxylate in plasma would interfere with the oxalate assay. In fact, it has been
310
confirmed here, that glyoxylate does not itself interfere with the oxalate assay, when in aqueous solution free from enzymes, as previously reported [9]. On the other hand, when glyoxylate was added to plasma, the apparent oxalate level was slightly increased, and when added to whole blood, the level was enormously increased (Table III). Xanthine oxidase can be inhibited by allopurinol [17], LDH by borate [ 181 and glycollate oxidase by phenyllactate [ 191, and it was, therefore, decided to try the effect of adding a mixture of these three inhibitors to the syringe used for venepuncture. Table II shows that this addition resulted in a considerable fall in the apparent oxalate level, although it had no effect on the assay of oxalate in aqueous solutions. It therefore seems reasonable to suppose that the inhibitors were suppressing formation of oxalate in plasma or blood in vitro and that the probable causeof such additional oxalate was glyoxylate. These results seem to show that no chemical method of measuring plasma oxalate at normal levels in vitro can be accurate, unless a method of blocking further formation of oxalate is used. It is therefore clear that all previous normal figures quoted for in vitro determination of oxalate are too high, and it seems likely that the lower in vivo isotopic determinations are correct. The normal values now reported are the lowest yet obtained by an in vitro method and they are only slightly higher than those found by in vivo isotopic methods [l]. It is possible that even these are too high, as we cannot be certain that inhibition of oxidation of glyoxylate was always complete. It is not easy to ensure instantaneous mixing of inhibitor and blood in the syringe and there could be other reasons as well. Measurements of oxalate at such low levels as now reported in normal plasma are at the limits of sensitivity of the method and are not as accurate as one would like. It remains to be explained why in chronic renal failure the in vivo and in vitro methods appear to agree [4]. It is known that LDH activity in uraemic serum is reduced by dialysable substances, thought to be urea and oxalate [20], and oxalate itself inhibits the oxidation of glyoxylate to oxalate [lo]. As it seems that both plasma urea and oxalate are greatly increased in chronic renal failure, it may well be that the spontaneous oxidation of glyoxylate to oxalate does not take place in the blood of patients with this condition. Furthermore, since the plasma oxalate is so much higher in chronic renal failure the effect of oxidation of glyoxylate to oxalate would be much less apparent. It should be pointed out that the measurement of plasma oxalate by the method described is highly demanding, tedious, and requires a large volume of blood. It is, therefore, not recommended for routine laboratory use, but does seem to have a place in research projects such as that described here. Acknowledgements We are grateful to Wellcome Laboratories Ltd. for the gift of Allopurinol powder, and to the normal volunteers who donated their blood so freely.
311
References 1
Hodgkinson,
2
Williams,
3
Hodgkinson.
4
Constable, Med.
5
Knowles, Krugers
(1977)
Oxalic
Johnson. A.
Acid
G.A.
and
and Wilkinson,
R.
A.R..
56.
6
A. H.E.,
Joekes.
A.M.,
in Biology
and
Medicine.
Smith,
L.H.
(1971)
(1974)
Clin.
Sci.
Kasidas.
G.P..
Academic
Clin. Molec.
O’Regan,
Press,
Sci.
41.
213-218
Med.
46,
61-73
P.
and
Rose,
G.A.
London
(1979)
Clin.
Sci.
Molec.
299-304 C.F.
and
Hodgkinson,
Dagneaux,
A.
P.G.L.C..
(1972)
Klein
Analyst
Elhorst,
97,474481
J.T.
and
Olthuis,
F.M.F.G.
(1976)
Clin.
Chim.
Acta
71.
319-325 7
Hatch,
M.,
8
Nuret,
P. and
Bourk,
9
Hallson,
P.C.
10
Fisher.
V.
11
Sallis.
d.D.,
12
Hockaday,
E. and
Offner. and
Rose.
and Watts,
(1978) G.A.
R.W.E.
Lumley, T.D.R.,
Costello,
M.
M.F.
J. (1977) Clin.
Chim.
Clin.
Chem.
Acta
82.
9-12
Acta
55.
(1974)
Clin.
Chim.
(1968)
Clin.
Sci.
and
Jordan.
Frederick,
E.W.,
J.E.
34,
76-78
29-39
97-110
(1977)
Clayton,
23,
Biochem.
J.E.
and
Med.
Smith,
18.
L.H.
371-377
(1965)
J. Lab.
Clin.
Med.
65,
677-
687 13
Kasidas,
14
Booth,
G.P.
15
Banner,
16
Richardson,
17
Elion,
18
Nachlas,
19
Liao,
20
Wilkinson.
V.H.
and
M.R.
L.L.
and K.E.
B.G.
Rose,
(1938)
Rosalki, and
(1966)
G.A.
Ann.
Margulies,
and
Richardson, Fujimoto.
Clin.
J. 32.
S.B.
Tolbert,
M.M., J.H.,
(1979)
Biochem.
Rheum. S.I.. K.E. Y.,
(1967) N.E.
Chin
Acta
96,
25-36
494-502 Nature
(1961)
Dis.
25,
Goldberg. (1973) Senesky,
213,
J. Biol.
726-727 Chem.
236.
1280-1284
608 J.D. Arch. D. and
and
Seligman,
Biochem. Ludwig,
M.A.
Biophys. G.D.
(1960)
Anal.
Biochem.
1, 317-326
154,68-75
(1970)
J. Lab.
Clin.
Med.
75,
109