urinary alkoxyacetic acids and effects exposure to …pathway is also interesting because...

Occupational and Environmental Medicine 1996;53:595-600

Urinary alkoxyacetic acids and renal effects ofexposure to ethylene glycol ethers

J Laitinen, J Liesivuori, H Savolainen

AbstractObjectives-Ethylene glycol ethers andtheir acetates are widely used in industry,because of their hydrophilic and simulta-neously lipophilic properties. Ethyleneglycol ethers and their acetates aremainly metabolised to alkoxyacetic acids,but there is also a minor pathway throughethylene glycol to oxalic acid. The mainpathway of ethylene glycol ethers is asso-ciated with significant clinical or experi-mental health effects and the minorpathway is also interesting because for-mation of urinary stones depends princi-pally upon the urinary concentration ofoxalate and calcium.Methods-Excretion of alkoxyacetic andoxalic acids was examined amongsilkscreen printers for an entire workingweek. The aim of the study was to evalu-ate alkoxyacetic acids as early indicatorsof exposure to glycol ethers and to evalu-ate their toxicity to kidneys. The load ofalkoxyacetic and oxalic acids was com-pared with the excretion ofcalcium, chlo-ride, ammonia, and glycosaminoglycans(GAG). Morning urine was chosen for themain analysis, as the overall metabolite,ethoxyacetic acid (EAA), has a long elim-ination time from the body.Results-The excretion of calciumincreased according to the urinaryalkoxyacetic acid load. The excretion ofammonia and chloride was higher among

Ethylene glycol

N-Alkoxyacetylglycine

Glycol aldehyde

Glycolic acid

1 > ~~Glyoxal

Glyoxylic acid

| \s ~ Formic acid

Oxalic acid Glycine

Figure 1 Metabolic pathway ofalkoxyethanol acetate.24

Alkoxyethanol acetate

Alkoxyethanol

Alkoxyacetaldehyde

Alkoxyacetic acid

the exposed workers than among the con-trols. The highest urinary alkoxyaceticacid load was also associated withincreased excretion of GAG, which mayreflect the toxicity of metabolites of ethyl-ene glycol ether. The excretion of GAGcorrelated positively with that of calciumin the printers with highest exposure. Thetendency to form urinary stones was 2-4-fold higher among silkscreen printersthan among office workers.Conclusion-On the basis of renal effectsour study indicates the need for establish-ing a new biological exposure limit beforea workshift that is clearly below 100 mmolethoxyacetic acids per mol creatine inmorning urine of people occupationallyexposed to ethylene glycol ethers.

(Occup Enznron Med 1996;53:595-600)

Keywords: metabolic acidosis; oxalic acid; urinarystones

Ethylene glycol ethers and their acetates arewidely used in industry, because of theirhydrophilic and simultaneously lipophilicproperties. Glycol ethers do not have specialwarning properties-for example, odour-andthey rapidly enter the body unnoticed. Glycolethers have low vapour pressure, but they arereadily absorbed through the skin.)

Ethylene glycol ethers and their acetates aremainly (60%-80%) metabolised throughalkoxyacetaldehydes to alkoxyacetic acids (fig1). There is also a minor (10%-15%) pathway(fig 1) through ethylene glycol, glycolalde-hyde, glycolic acid, and glyoxylic acid to oxalicacid.24The main pathway of ethylene glycol ethers

through acetaldehyde to alkoxyacetic acids isassociated with significant clinical or experi-mental health effects. Exposure to ethyleneglycol ethers is associated with haematologicalproblems,56 oligospermia, azoospermia,7 andreproductive problems.8 Animal studies indi-cate that they can induce germ cell toxicity,reproductive toxicity, and teratogenicity, andsome of them are potential carcinogens.9The minor pathway is also interesting

because formation of urinary stones dependsprincipally upon the urinary concentration ofoxalate and calcium, although the percentageof oxalate in stones is higher than that of cal-cium.10 On the other hand, calcium oxalatenephrolithiasis is associated with a defect inerythrocyte oxalate self exchange and an

Kuopio RegionalInstitute ofOccupational Health,Kuopio, FinlandJ LaitinenJ LiesivuoriInstitute ofOccupational HealthSciences, University ofLausanne, Lausanne,SwitzerlandH SavolainenCorrespondence to:Dr Juha Laitinen, KuopioRegional Institute ofOccupational Health,PO Box 93, FIN-70701Kuopio, Finland.Accepted 26 March 1996

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abnormal rate of phosphorylation of erythro-cyte membrane protein." The glycosamino-glycan (GAG) content of erythrocyte ghosts isfound to be lower than normal in patients whoform stones. The GAG content correlatesinversely with oxalate self exchange and band3 phosphorylation in erythrocytes. The ery-throcyte oxalate is transported by way of band3 protein, which shares the same functionalsimilarities with anion exchangers, but it is notknown whether non-erythroid anion exchang-ers are structurally related to it.'2 Anyway, therole of glycosaminoglycans as inhibitors of cal-cium oxalate crystallisation is controversial,and reported actions range from neglible" andinhibitory'4 to promoting.'5

Karniski and Aronson have suggested thatthere are at least two separate anion exchangersmediating Cl- transport on the luminal mem-brane of the rabbit proximal tubule cell. Theseexchangers may play important parts in medi-ating transtubular Cl- and oxalate transport inthis nephron segment.'6 17 The proximal tubularcells also liberate ammonia from glutamineand the glutamate pool. It is possible that ahigher oxalate concentration in these cellscould also cause changes in the liberation ofNH4+ in humans.'8

According to these findings, the excretion oflarge amounts of organic acids poses problemsto renal tubular function, and the interferenceseems to change the function of the chlorideexchanger in the kidney.'9-21From a clinical point of view in occupa-

tional health care, the kidney effects are proba-bly the most interesting as most of the glycolether metabolites are excreted in the urine.Therefore, additional knowledge on the possi-bility of a link between exposure to ethyleneglycol ether and changes in renal functionswould be advantageous. More information on

the minor pathway of metabolism would alsobe important, because small changes inoxalate excretion have a greater impact on thesaturation of calcium oxalate in urine thanchanges in calcium excretion.2223 The changesin the excretion of proteoglycans may be sec-

ondary, but they may reflect more chronicchanges in the kidney. All variables are valu-able with a view to validating the current bio-logical exposure indices for ethylene glycolethers and their congeners.

MethodsEight silkscreen printers from four worksitesparticipated in this study on a voluntary basis.Their ages varied from 20 to 40 years and they

were all considered to be healthy. Urine sam-ples were collected before and after the work-shift in 56 instances and analysed foralkoxyacetic and oxalic acids, ammonia, cal-cium, and pH. Urinary analysis before a work-shift was completed with assay of GAGs andchloride (table 1). Twenty one office workerswho were not exposed to ethylene glycol etherswere the control group. They were consideredhealthy and they gave urine samples beforetheir workshift. Samples were not included inthe study if the concentration of creatinine wasbelow 2 mmol/l.

All of the collected urine samples were keptat - 20°C before the analysis, and thoseobtained for the oxalic acid analysis were notstored with preservatives. Methoxyacetic acid(MAA), ethoxyacetic acid (EAA), butoxyaceticacid (BAA), and oxalic acid were analysed withthe modified gas chromatographic methoddescribed by Sakai et al.24 The coefficients ofvariation within assays (n = 5) for MAA,EAA, BAA, and OA were 9-2%, 2-6%, 1-4%,and 3A4%, respectively, at a concentration of20 mg/l. Calcium was analysed with a spec-trophotometer,25 ammonia with a reflectrome-ter26 and GAGs by the method of Savolainen.27The workers were divided into four groups

according to the alkoxyacetic acid burden. Thepurpose of the study was to evaluate the dose-response relation between the exposure andexcretion. The first group (> 100 mmolalkoxyacetic acid/mol creatinine) and secondgroup (100-50 mmol/mol creatinine) were

formed with two international limit values forEAA, 1 10 mmol/mol creatinine (AmericanConference of Governmental IndustrialHygienists (ACGIH) 1994-5),28 and 55mmol/mol creatinine (Deutsche Forschung-sgemeinschaft (DFG) 1994).29 Limits for thethird and fourth groups were chosen to be50-20 mmollmol and 20-0 mmol alkoxyaceticacid per mol creatinine. The limit value forEAA was chosen, because the portion ofMAA,EAA, and BAA of total excretion of alkoxy-acetic acids were on average 1-0%, 93-6%, and5 4%, respectively.The significance of the difference in the

excretion of oxalic acid, calcium, ammonia,chloride, pH, and GAG between the exposedand unexposed groups was tested by analysis ofvariance (ANOVA). The results for theexposed group were repeated observationsfrom the same set of people and for the unex-

posed group they were single values. The sta-tistical differences between exposed andcontrol groups of the excretions and excretionpattern were tested separately each day. The

Table 1 Mean (SEM) excretions of silkscreen printers after the shift compared with those of controls

Silkscreen printersControls Monday Tuesday Wednesday Thursday Friday Saturday Sunday

AAA (mmol/mol creatiine) ND 44 (18) 71 (30) 77 (22) 87 (27) 94 (35) 59 (23) 46 (17)Oxalic acid (mmol/mol creatine) 38 (3) 49 (11) 43 (5) 36 (4) 53 (8)t 54 (15) 55 (12)t 47 (6)Calcium (mmol/mol creatinine) 243 (30) 211 (37) 217 (43) 176 (39) 193 (22) 261 (62) 320 (69) 233 (47)Ammonia (mol/mol creatimine) 2-8 (0 6) 3-6 (0-5) 3-2 (0-5) 4-0 (0-7) 4-0 (0-5) 3-7 (0-3) 4-0 (0-3) 3-3 (0-3)pH 6-4 (0-3) 6-0 (0-3) 6-3 (0-2) 5-8 (0-2) 5-8 (0-2)t 6-1 (0-2) 5-7 (0-2)t 5-9 (0-2)P value (mva) - 0-2505 0-9389 0 0864t 0.0357* 0-2787 0-0899* 0-1912

*P<0-05; -P-<0-1.ND = not detected; AAA = alkoxyacetic acids; mva = multivariance analysis.

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Urinary alkoxyacetic acids and renal effects of exposure to ethylene glycol ethers

Table 2 Mean (SEM) excretions of silkscreen printers before shifts compared with those of controls

Silkscreen printers

Controls Monday Tuesday Wednesday Thursday Friday Saturday Sunday

AAA (mmol/mol creatinine) ND 30 (15) 27 (9) 70 (31) 88 (39) 82 (26) 87 (36) 45 (20)Oxalic acid (mmol/mol creatinine) 38 (3) 36 (3) 32 (4) 42 (4) 42 (4) 41 (6) 34 (3) 35 (7)Calcium (mmol/mol creatinine) 243 (30) 437 (87)* 332 (73) 373 (77)t 452 (105)* 337 (51) 291 (30) 166 (35)Ammonia (mol/mol creatinine) 2-8 (0-6) 4-0 (0-5)t 4-7 (0 8)t 4-7 (0-8)t 4-9 (0 5)* 4 4 (0-6)t 3 5 (0-3) 3-8 (0-3)pH 6-4 (0-3) 5-9 (0-2) 5-7 (0-1)t 6-0 (0-2) 6-0 (0-2) 5-7 (0-2) 5-7 (0-2)t 5-5 (0 1)*P value (mva) - 0-2312 0-1993 0-3135 0-0699t 0-1916 0-4658 0.0129*GAG (g/mol creatinine) 4-7 (0-5) 4-4 (1-1) 4-5 (1-0) 4-3 (0-7) 7-0 (1-5) 4-5 (0-8) 4-8 (1-4) 2-9 (0-8)tChloride (mol/mol creatinine) 10-9 (1-3) 17-3 (4-1)t 13-8 (1-6) 13-3 (2-2) 19-3 (6-5) 16-7 (3-2)t 20-0 (6) 9-2 (1-7)

*P < 0 05d d G< 0 1.ND = not detected; GAG = glycosaminoglycans; AAA =alkoxyaceiic acids; mva = muliivaniance analysis.

significance of the difference in the excretionpatterns between exposed and control groupswere tested with multivariance analysis, whichincluded oxalic acid, calcium, ammonia, andpH, because they had same control group.The tendency (T) for subjects to form

stones was estimated by calculating the short-est distance (D) of calcium (= x1) and oxalicacid (= y,) values from the supersaturationcurve {x, (3655907,93a- 097)(x -97)} by a com-puter program (Mable V, Waterloo MableSoftware, Waterloo, Ontario, Canada) forsymbolic mathematics. For each subject a satu-ration curve was corrected according to theexcretion of creatinine (a). Distances were

420

a)'E 380a)

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-3 340E0E 300EE.2 260

220

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5

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343325 ~~~(65)

(40)

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Control 7(1) 31(3) 68(4) 185(19)

Alkoxyacetic acids (mmol/mol creatinine)

4.8C (0.5)

4.4

(0.2)

4.1

(0.4) 3.9Control 7(1) 313) 68(4 185(0.2)

(0.6)H

Control 7(1) 31(3) 68(4) 185(19)

marked negative for subjects who exceeded thesaturation point (x, > x). The T was calculatedby the equation T = 1/D(mean).30

ResultsThe results were separated into urine analysesbefore and after the shift. The analysis after theshift did not show any dose-response relationbetween the alkoxyacetic acid burden and theexcretion of calcium, ammonium, or the pH.The urinary excretion of alkoxyacetic acidsincreased during the working week and thehighest levels of excretion occurred on Fridayevening. The highest peak of oxalic acid was

6

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4.5

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4.7(0.5)

4.6(0.5)

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Control 7(1) 31(3) 68(4) 185(19)Alkoxyacetic acids (mmol/mol creatinine)

20 r

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(1.3)

Control 7(1) 31(3) 68(4) 185(19)Alkoxyacetic acids (mmol/mol creatinine)

Figure 2 Relation between the excretion of alkoxyacetic acids and that of calcium, GAG, ammonia and chloide. Theresults are given as the means (SEM).

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Figure 3 Relationbetween the excretion ofGAG and calcium inthe most exposed group(y = 120174x + 48-34,r= 075).

0)c._

am-

o-.

0

S

0 300 600 900 1200Calcium (mmol/mol creatine)

also found at the end of the week. The excre-tion of oxalic acid and pH differed moderatelyfrom controls on Thursday and Saturday. Theexcretion patterns of exposed workers differedmoderately from those of the control group onWednesday and Saturday and significantly onThursday evening (table 1).

Urinary ammonia, calcium, or pH before theshift differed significantly from those of controls

A

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on Monday, Thursday, and Sunday mornings(table 2), and the excretion of calcium seemedto follow the alkoxyacetic acid burden (fig 2).The excretion of chloride was higher among theexposed workers than among the controls (fig2). The excretion patterns of exposed workersdiffered moderately from those of controls onThursday and significantly on Sunday morning(table 2).The mean excretion of GAG seemed to

decrease from the control group to the secondmost highly exposed group but the mean excre-tion in the most highly exposed group was byfar the greatest (fig 2). The excretion of gly-cosaminoglycans (y) correlated positively(y = 120 74x + 48-34, r = 0 75) with that ofcalcium (x) in this group (fig 3).The calculated distance (mean (SEM)) from

the supersaturation curve were amongunexposed workers, silkscreen printers beforethe shift, and silkscreen printer after the shift(149 (20), 62 (9), and 104 (18) respectively).The mean tendencies to form urinary stoneswere 0-006, 0-016, and 0-010, respectively.

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Figure 4 (A) Unmodified supersaturation curvefor calcium oxalate,3' (B) modified supersaturation curvefor controlsamples according to the mean excretion ofcreatinine (11 13 mmol/l), (C) modified curvefor samplesfrom silkscreenprinters before a workshift according to the mean excretion of creatinine (14 20 mmol/l), and (D) modified curveforsamples from silkscreen printers after a workshift according to the mean excretion ofcreatinine (1255 mmolli). All cases inthe different groups have been plotted into the figures and results of silkscreen printers are comparable with the same scaled

results of the controls.

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Urinaty alkoxyacetic acids and renal effects ofexposure to ethylene glycol ethers

DiscussionThe urinary analysis after the shift did notshow any significant differences between theexposed and unexposed workers (table 1).This lack of correlation may have been causedby the long biological half life of the metabo-lites in the main metabolic pathway. The halflife of alkoxyacetic acids decreases as thelength of the alkyl chain increases. The halflives for MAA, EAA, and BAA are 71, 42, andsix hours, respectively.2 31-33 This could alsocause a metabolic adaptation to the generationof the organic acid metabolites in kidneys.Most of MAA, EAA, and BAA is not typicallyconjugated with glycine.3 Therefore, a signifi-cant reabsorption probably occurs also in thetubular section of the nephron. The liberationof ethylene glycol represents the minor meta-bolic pathway of ethylene glycol ethers. Thehalf life of ethylene glycol is about three toeight hours and the highest peak occurs inblood one to four hours after exposure.34Therefore, it seems that the end metabolite ofethylene glycol, oxalic acid, does not accumu-late during a working week when the doses ofethylene glycol ethers are moderate.A formate and chloride exchanger protein

has been isolated from the kidney membranefraction.35 It may also accept anions other thanformate or oxalate in the exchange system, asin our study the urinary chloride concentrationfollowed that of acids (fig 2). The exchangerwas inhibited by furosemide, and one wonderswhat effects its use would have on the kineticsof alkoxyacetic acids. In one case of intoxica-tion, a dose of furosemide helped the victim tosurvive a very high dose of formic acid.36

It is interesting to note that the urinaryammonia increased with increasing urinaryalkoxyacetic acid (fig 2). It is possible thatalkoxyacetic acids change the renal ammonia-genesis at high concentrations as does formicacid.20 As ammonia occurs in the urine pri-marily as NH4+ ion, its excretion would requirethe excretion of an equivalent number ofanions such as chloride or phosphate. Thislink was also found in our study (fig 2). Freeammonia is very toxic and it cannot be trans-ported through the blood; therefore it is con-verted into to an amide group of glutamine byglutamine synthetase.The highest urinary acid load is also associ-

ated with increased proteoglycan excretion,which may also reflect the toxicity of the glycolether metabolites (fig 2). The excretion ofGAG seems to decrease with the load ofalkoxyacetic acids in moderate exposure aswas found to be the case in our previous studyamong car mechanics.37 Baggio et al also founda lower GAG content in erythrocyte ghosts inpatients who form stones than in normal peo-ple."I

Calciuria could also qualify as a renal effectindicator because of the associated risk ofnephrolithiasis. It is interesting to note that theproteoglycan excretion peak coincided withthe highest rate of calcium excretion. It hasbeen maintained that urinary proteoglycansprevent the crystallisation of urinary calciumsalts.38 The excretion of calcium increased

with increasing alkoxyacetic acid. This occur-rence in turn is positively correlated with thatof GAG (fig 3). The increase in urinary GAGmay explain the finding that none of our caseshad a history of urinary stone disease despitethe fact that the urinary calcium and oxalateconcentrations were at or above the supersatu-ration line in many cases.

Formation of urinary stones starts rapidlywhen the products of the reaction of oxalicacid with calcium exceeds the supersaturationlevel. The tendency to form crystals in urinesamples can be estimated roughly by hyper-bolic function30 (fig 4). Urine samples takenbefore the shift showed a higher tendency toform urinary stones than did samples takenafter the shift. The calculated results indicateda 2-4-fold higher tendency for urinary stonesamong silkscreen printers than among officeworkers in samples taken before a shift (fig 4).

Dietary oxalate is thought to contribute10% to 20% to urinary oxalate, 40% to 50%to endogenous metabolism, and the remaining40% to 50% to the breakdown of ascorbic acidin the body.39 It is well known that hormonalfluctuation may contribute more to the vari-ability in oxalate excretion than for examplethe dietary intake of protein.40 A good exampleof a hormonal effect is the fact that womenexcrete more oxalate relative to creatinine thanmen. The prevention of urolithiasis through adecreased dietary intake of calcium and oxalicacid is controversial because a decreasedintake of calcium and oxalic acid increases theintestinal absorption and urinary concentra-tion of calcium and oxalic acid.4' The role ofexposure has been found to have a causal rela-tion to urolithiasis among railway shopmen,4'and also in occupational groups with lowphysical activity. A similar relation has alsobeen found with exposure to high tempera-tures and an increased fluid loss.43

In conclusion, chronic occupational expo-sure to ethylene glycol ethers and their con-geners is associated with important alkoxy andoxalic acids loads which alter renal function.The risk of urinary stones seems to be higheramong silkscreen printers than among con-trols, and some of the changes are typical ofchronic metabolic acidosis. It is very impor-tant to emphasise that ethylene glycol ethersare well absorbed through the skin and thatanalyses for their vapours mostly underesti-mate the exposure. Metabolites of ethyleneglycol ethers also accumulate during the work-ing week so that biological monitoring is theonly means of obtaining an evaluation of totalexposure. Our study also indicates the need,on the basis of renal effects, to establish a newbiological exposure limit clearly below 100mmol ethoxyacetic acids per mol creatinine, inthe morning urine before a workshift, of peo-ple occupationally exposed to ethylene glycolethers.

We thank Ms N Chavannes, Mrs T Turunen, and Mr J Marinfor their excellent technical assistance and the Finnish WorkEnvironment Fund for its financial aid.

1 Angerer J, Lichterbeck E, Begerow J, Jekel S, Lehnert G.Occupational chronic exposure to organic solvents. IntArch Environ Health 1990;62:123-6.

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urinary alkoxyacetic acids and effects exposure to …pathway is also interesting because...

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