The composition of four-hour urine samples from patients with calcium oxalate stone disease

Urine collected during a 24-h period between 06.00 and 10.00 h from 25 patients with recurrent CaOx stone disease was analysed with respect to calcium, oxalate, magnesium, citrate and creatinine. Urinary excretion of oxalate in relation to creatinine was slightly higher in 24-h urine but the correlation between 24-h and 4-h values was good. Good correlations were also recorded for calcium and citrate, whereas a more variable result was obtained for magnesium. In terms of the risk of forming a supersaturated urine (CaOx risk index), a good correlation was observed between 24-h and 4-h urine samples, although the highest values were found in 24-h urine. As a result of a low mean urine flow between 06.00 and 10.00 h, the highest supersaturation in terms of the AP (CaOx) index was observed in these samples. When the risk of calcium oxalate crystallisation (CaOx-CR) was determined by means of the increment in oxalate concentration required for precipitation of CaOx, 7 of 11 samples had the highest values in the 4-h urine. Samples collected during a 4-h period might thus be useful in the evaluation and follow-up of CaOx stone formers and further studies will show to what extent they can replace 24-h urine collections.     

A nidus,crystalluria and aggregation: key ingredients for stone enlargement

The in vitro study of calcium oxalate (CaOx) stone formation is usually based on crystallisation models but it is recognised that both healthy individuals and stone formers have crystalluria. We have established a robust in vitro stone growth model based on the principle of mixed suspension, mixed product removal system (MSMPR). Utilising this technique we studied the influence of CaOx crystallisation kinetics and the variation of calcium and oxalate concentrations on CaOx stone growth in vitro. Six stones received standard concentration of Ca (6 mM) and Ox (1.2 mM) in the medium while another six received variable concentrations of both Ca and Ox at various intervals. Stone mass was plotted against the experiment duration (typically 5–7 weeks). The stone growth was dependent on sufficient input calcium and oxalate concentrations and once triggered, stone growth could not be maintained at reduced calcium and oxalate inputs. The stone growth rate was positively correlated to the number of crystals in suspension around the stone and to the crystal nucleation rate and negatively correlated to the crystal growth rates. This leads to the conclusion that aggregation of crystals from the surrounding suspension was the dominant mechanism for stone enlargement.     

Relationship between supersaturation and calcium oxalate crystallization in normals and idiopathic calcium oxalate stone formers

BACKGROUND: In an earlier study on recurrent CaOx stone formers with no detectable abnormalities, we found that the urine of these subjects had a lower tolerance to oxalate load than controls and that the removal of urinary macromolecules with a molecular weight greater than 10,000 D improved their tolerance to oxalate. METHODS: The effects on CaOx crystallization of reduced urinary supersaturation of calcium oxalate (CaOx), induced by night water load, were studied in 12 normal males and in 15 male OxCa stone formers who were free from urinary metabolic abnormalities. The effect of the macromolecules, purified and retrieved from the natural and diluted urine, were analyzed in a metastable solution of CaOx. RESULTS: The water load caused an increase in urine volume (from 307 +/- 111 to 572 +/- 322 ml/8 hr, P = 0.014 in normal subjects, and from 266 +/- 92 to 518 +/- 208 ml/8 hr, P = 0.001 in the stone formers) and a concomitant reduction of the relative CaOx supersaturation (from 8.7 +/- 2.5 to 5.1 +/- 2.5 ml/8 hr, P = 0.001 in normal subjects, and from 10.4 +/- 3.5 to 5.0 +/- 2.7 ml/8 hr, P = 0.001 in the stone formers). The decrease in CaOx supersaturation was accompanied by an increase of the permissible increment in oxalate, both in normal subjects (from 43.8 +/- 10.1 to 67.2 +/- 30. 3 mg/liter, P = 0.018) and in the stone formers (from 25.7 +/- 9.4 to 43.7 +/- 17.1 mg/liter, P = 0.0001), without any significant variations of the upper limit of metastability for CaOx (from 21.6 +/- 5.3 to 20.5 +/- 4.2 mg/liter in normal subjects, and from 18.7 +/- 4.5 to 17.1 +/- 3.7 mg/liter in the stone formers). The inhibitory effect of urinary macromolecules with molecular weight greater than 10,000 Daltons did not undergo any change when the latter were recovered from concentrated or diluted urine, either in normal subjects or in the stone formers. CONCLUSIONS: Reduced CaOx supersaturation by means of water load has a protective effect with regards to CaOx crystallization in subjects who do not present any of the common urinary stone risk factors.     

Renal oxalate excretion following oral oxalate load in patients with urinary calculus disease and healthy controls

Oral oxalate loading using sodium oxalate or a vegetable juice was done to evaluate the intestinal absorption of exogenous oxalate in 30 patients with renal stones and 13 healthy controls. Fifteen calcium oxalate stone formers, 7 non-oxalate stone formers and 10 healthy volunteers were given an oral loading of sodium oxalate (500 mg). Urinary oxalate increased promptly, reaching a peak value within 4 to 8 hours after administration of a synthetic oxalate orally in a fasting state. In calcium oxalate stone formers, the mean increment of urinary oxalate and the bioavailability following oral sodium oxalate load were significantly higher than in the healthy controls and non-oxalate stone formers. Furthermore, intestinal hyperabsorption of oxalate in our criterion was defined in six patients with calcium oxalate stones (40%). On the other hand, eight calcium oxalate stone formers and three healthy controls were given vegetable juice. Urinary oxalate was increased only slightly after the ingestion, and there was no difference between calcium oxalate stone formers and normal controls. These results suggest that a certain hyperoxaluria might be induced by intestinal absorption of exogenous oxalate, and that the hyperabsorption might indicate a possible risk factor for calcium oxalate stone formation.     

Oxalate loading test for outpatients with calcium oxalate stones

A spinach loading experiment was performed on 9 normal subjects, 25 outpatients who were single calcium oxalate stone formers and 25 recurrent calcium oxalate stone formers. The experimental diet contained 445 mg of total oxalate, 163 mg of soluble oxalate and 115 mg of calcium. Urinary oxalate excretion was observed 2 hrs before and 6 hrs after the experimental diet was consumed. There was no significant difference in urinary oxalate excretion in preloading urine of normal subjects and stone formers. However, urinary oxalate excretion in postloading urine was significantly elevated in stone formers. This loading test is recommended as a simple and valuable screening method of hyperabsorption of oxalate on outpatients with calcium oxalate stones.     

Dual roles of brushite crystals in calcium oxalate crystallization provide physicochemical mechanisms underlying renal stone formation

Calcium oxalate monohydrate (COM) crystals are the major mineral component of most kidney stones, and thus have an important role in chronic human disease. However, the physicochemical mechanisms leading to calcium oxalate (CaOx) stone disease are only partially defined. As spontaneous precipitation of CaOx is rare under renal conditions, an alternative pathway for CaOx crystallization seems necessary to resolve this central issue. We performed kinetic studies using the dual constant composition method to simultaneously analyze the crystallization of COM and brushite, the form of calcium phosphate that is most readily formed in the typical slightly acidic urinary milieu. These studies were supported by parallel analysis by scanning electron and atomic force microscopy. In these studies, mineralization of a thermodynamically stable phase (COM) was induced by the presence of brushite, a more readily precipitated inorganic phase. Furthermore, once formed, the COM crystals grew at the expense of brushite crystals causing the dissolution of the brushite crystals. These studies show that brushite may play crucial roles in the formation of COM crystals. The definition of these two roles for brushite thereby provides physicochemical explanations for the initiation of COM crystallization and also for the relative paucity of calcium phosphate detected in the majority of CaOx renal stones.     

Intestinal Oxalobacter formigenes colonization in calcium oxalate stone formers and its relation to urinary oxalate

BACKGROUND AND PURPOSE: Oxalobacter formigenes is an anaerobic commensal colonic bacterium capable of degrading oxalate through the enzyme oxalyl-CoA decarboxylase. It has been theorized that individuals who lack this bacterium have higher intestinal oxalate absorption, leading to a higher urinary oxalate concentration and an increased risk of calcium oxalate urolithiasis. We performed a prospective, controlled study to evaluate O. formigenes colonization in calcium oxalate stone formers and to correlate colonization with urinary oxalate and other standard urinary stone risk factors. PATIENTS AND METHODS: Thirty-five first-time calcium oxalate stone formers were compared with 10 control subjects having no history of urolithiasis and a normal renal ultrasound scan. All subjects underwent standard metabolic testing by submitting serum and 24-hour urine specimens. In addition, all subjects submitted stool samples for culture and detection of O. formigenes by Xentr(ix) O. formigenes Monitor. RESULTS: Intestinal Oxalobacter was detected in only 26% of the stone formers compared with 60% of the controls (p < 0.05). Overall, the average urinary oxalate excretion by the two groups was similar (38.6 mg/day v 40.8 mg/day). Among stone formers, however, there were statistically higher urinary oxalate concentrations in O. formigenes-negative patients compared with those testing positive (41.7 mg/day v 29.4 mg/day) (p = 0.03). Furthermore, all 10 stone formers with hyperoxaluria (>44 mg/day) tested negative for O. formigenes (p < 0.05). CONCLUSIONS: Calcium oxalate stone formers have a low rate of colonization with O. formigenes. Among stone formers, absence of intestinal Oxalobacter correlates with higher urinary oxalate concentration and an increased risk of hyperoxaluria. Introduction of the Oxalobacter bacterium or an analog of its enzyme oxalyl-CoA decarboxylase into the intestinal tract may be a treatment for calcium oxalate stone disease.     

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in␣vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine. Received: 24 July 1997 / Accepted: 2 January 1998     

Effects of urinary pH and acid-base balance on the formation of calcium oxalate stone

The effects of urinary pH and acid-base balance on the calcium oxalate stone formation was investigated by two experiments. 24-hr urine samples were collected from 15 recurrent CaOx stone formers, 9 single stone formers and 6 age-matched controls. Inhibitory effect of 1% urine in various pH (4.0-9.0) were calculated by a seed crystal method. In the seed crystal system, there were no significant differences in the inhibitory activity of aggregation (Ia) and in the inhibitory activity of size (Is) for each pH of metastable solution between the stone former group and the control group. However, the value of Ia and Is showed a tendency of rise in proportion to a rise in pH. Rats model for calcium oxalate urolithiasis were fed with three different diets (1% NH4Cl, 5% NaHCO3 and 8% NaHCO3 diet) for three weeks. On the fourth week, 24-hr urine samples were collected. In the animal experiment, calcium oxalate stone formations were predominantly recognized in the kidney of the 1% NH4Cl diet group. The biochemical data showed an increase of urinary calcium and oxalate, and a decrease of urinary citrate. These results suggest that low urinary pH and metabolic acidosis are promoters of the calcium oxalate stone formation.     

Quantification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in vitro in the urine of healthy controls and stone-forming patients.

We have previously identified proteins extracted from calcium oxalate (CaOx) and calcium phosphate (CaP) crystals generated experimentally in vitro in whole urine of healthy controls and stone formers. No significant differences were detected between protein components in matrices of crystals obtained from both groups. The aim of the present study was to estimate the amounts of six proteins identified earlier in order to investigate the differences, if any, between healthy controls and lithiasis patients. CaOx and CaP crystals were generated in the urine samples by adding an oxalate and phosphate load, respectively. Crystals were harvested, washed, dried, and analyzed. Crystal matrix protein was extracted by demineralizing crystals with EDTA solution, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and identified immunochemically using Western blot analysis. The quantity of each protein was estimated by densitometric analysis. The predominant proteins found in organic matrices of CaOx crystals induced in the urine of healthy controls were prothrombin-related proteins followed by albumin and osteopontin. In matrices of CaP crystals, the principal proteins were Tamm-Horsfall protein followed by albumin, prothrombin-related proteins and osteopontin. However, when crystals were induced in the urine of stone formers, albumin was the major component of the organic matrix of both CaOx and CaP crystals. We concluded that a subset of urinary proteins is present in crystal matrix. Among them, albumin seems to play a crucial role in kidney stone formation.     

Retardation of calcium oxalate precipitation by glutamic-oxalacetic-transaminase activity

Summary It has been found that calcium oxalate stone formers have low UGOT activity compared to healthy individuals (controls). Urine from stone formers with no GOT activity and no effect on calcium oxalate precipitation was incubated with GOT for various periods. Subsequently calcium oxalate precipitation was decreased and found to be considerably retarded i.e., the pathological urine after the incubation acted in a way similar to that of normal urine. The yield of Glutamic-Oxalacetic Transaminase (GOT) activity is gultamic acid. It was shown that glutamic acid has a significant retardation effect on the precipitation of calcium oxalate stone formation. Therefore it may be suggested that GOT activity involved in glutamic acid creation in situ, has a role in kidney stone formation.     

Prevalence of hyperoxaluria in idiopathic calcium oxalate kidney stone disease

Urinary excretion of oxalate, calcium and urate has been investigated in 88 patients affected by idiopathic calcium oxalate stone disease and in 20 normal subjects. Of these ions, only oxalate was found significantly higher in stone formers. Defining hyperoxaluria as urinary oxalate excretion greater than 2 SD above normal, 50% of stone-forming people were found to be hyperoxaluric. When stone formers were classified in normo- and hyperoxaluric, the prevalence of hypercalciuria, hyperuricuria, family history of stone disease and recurrencies in stone formation was the same in both groups. It is concluded that hyperoxaluria is a frequent finding in finding in idiopathic calcium oxalate renal stone disease.     

Evaluation of urine composition and calcium salt crystallization properties in standardized volume-adjusted 12-h night urine from normal subjects and calcium oxalate stone formers

The volume of 12-h night urine from ten normal men (NM), ten normal women (NW) and 31 male calcium stone formers (SFM) was adjusted to 750 ml and analysed with respect to supersaturation with calcium oxalate (CaOx) and calcium phosphate (CaP), inhibition of CaOx crystal growth and aggregation, as well as the CaOx and CaP crystallization propensity. Concentrations of oxalate and glycosaminoglycans and AP(CaOx) index, an estimate of the CaOx ion-activity product, were higher and the concentration of citrate lower in NM than in NW. In SFM the directly assessed risk of CaOx crystallization was higher and the inhibition of CaOx crystal growth lower than in NM. There were no differences between the groups regarding inhibition of CaOx crystal growth by 74% dialysed urine or inhibition of CaOx crystal aggregation. SFM with mixed CaOxCaP stones had a higher concentration of phosphate and a higher AP(CaP) index at pH 7.0 than SFM with CaOx stones.     

Effects of different doses of alkaline citrate on urine composition and crystallization of calcium oxalate

Summary Prophylactic treatment with alkaline citrate in patients with recurrent calcium oxalate (CaOx) stone disease results in reduced CaOx supersaturation and increased urinary citrate. The effects of a single evening dose were compared with those of two and three daily doses in six recurrent CaOx stone formers with hypercalciuria, hypocitraturia or raised calcium/citrate quotients. While on a standardized hospital diet the patients were given 7.5 g (28 mmol) of sodium potassium citrate (URALYT-U) in one, two, and three doses. Fractional urine collections during 24 hours were analyzed for pH, composition, and crystallization risk (CR). All dosage regimens had favourable effects on urinary calcium, citrate, calcium/citrate quotients, and CaOx-CR. The most sustained effect was recorded with three divided doses. Single evening doses resulted in the most pronounced effects between 22.00–06.00 h, thereby counteracting the increased risk of CaOx crystallization during that period. In terms of 24 h urine composition the best effect was recorded with alkaline citrate administered three times daily, but because of the favourable response by a single evening dose between 22.00–06.00 h the assumption was made that this dosage regimen might be sufficient to reduce the risk of CaOx crystallization and stone formation. However, the validity of such an assumption can only be established by long-term clinical studies.     

Concentrated urine and diluted urine: the effects of citrate and magnesium on the crystallization of calcium oxalate induced in vitro by an oxalate load

Supplementation of certain calcium crystallization inhibitors, such as citrate and magnesium, and the dilution of urine with water are now considered consolidated practice for the prevention of calcium kidney stones. The aim of this study is to verify, using tried and true in vitro methods, whether the effect of these inhibitors can manifest itself in different ways depending on whether the urine is concentrated or diluted. Calcium oxalate crystallization was studied on 4-h urine of 20 male idiopathic calcium oxalate stone formers, first under low hydration conditions (non-diluted urine) and then under high hydration conditions (diluted urine). Both the diluted and the non-diluted urine samples were subjected to three types of load: (a) an oxalate concentration increment of 1.3 mmol/l only; (b) an oxalate concentration increment of 1.3 mmol/l with a citrate concentration increment of 1.56 mmol/l; (c) an oxalate concentration increment of 1.3 mmol/l with a magnesium concentration increment of 2.08 mmol/l. In non-diluted urine, the addition of the citrate and magnesium did not modify the crystallization parameters under study. In contrast, in the diluted urine the addition of the citrate and magnesium led to a reduction in the total quantity of crystals (equivalent to 35–45%) and their aggregates (equivalent to 30–40%); at the same time, there was an increase in the diameter of the monohydrate calcium oxalate crystals, which also underwent a morphological change. In conclusion, the inhibitory effects of citrate and magnesium on the crystallization of calcium oxalate do not manifest themselves in highly concentrated urine.     

Oxalate binding proteins in calcium oxalate nephrolithiasis

The existence of several oxalate specific binding proteins have been demonstrated in human and rat kidney. These occur in both cortical and medullary cells and are distributed mostly in the subcellular organelles. About 1/3 of the total cellular oxalate binding was localised in the inner mitochondrial membrane while the rest was in the nucleus. The purified mitochondrial oxalate binding protein (62 kDa) was composed, with a higher molar proportion, of basic amino acids, and could accumulate oxalate on incorporation into liposomes. In the nucleus, histone H_1B (27.5 kDa), nuclear membrane protein (68 kDa) and nuclear pore complex protein (205 kDa) were present with oxalate binding activities. In addition, a 45 kDa calcium oxalate binding protein was identified in most of the subcellular organelles. Both mitochondrial and nuclear oxalate binding proteins and calcium oxalate binding protein have shown the kinetic properties of specificity, saturability, pH and temperature dependency, energy of activation and inhibition by substrate analogues. All oxalate binding proteins were sensitive to the transport inhibitor 4-4 diisothiocyano stilbene-2–2 disulphonic acid (DIDS), which is known to interact with the lysine moiety of the proteins. Calcium oxalate monohydrate (COM) crystals adsorbed oxalate binding proteins from human and rat kidney, and oxalate binding proteins isolated from human kidney stone matrix also exhibited the above kinetic properties. In experimental hyperoxaluria, all of the renal oxalate binding proteins showed enhanced oxalate binding activity with increased protein concentration. This enhanced oxalate binding activity was also attributed to increased lipid peroxidation, which correlated positively, and to decreased thiol status, which correlated negatively. A positive correlation was observed between the lipid peroxidation and both the oxalate binding activity of the in vitro peroxidised subcellular organelles and the purified protein. Similarly, in an in vivo hyperoxaluric condition, a negative correlation was observed between thiol content and both the oxalate binding activity of the peroxidised subcellular organelles and the purified protein. In the calcium oxalate crystal growth system, the oxalate binding proteins behaved either as promoters or inhibitors of the nucleation and aggregation of crystals. Following the peroxidation of the proteins, the degree of effect of the promoter protein was further stimulated while the degree of inhibition caused by the inhibitor protein further declined. Similar observations were duplicated with the proteins derived from hyperoxaluric rat kidney or kidney homogenate subjected to in vitro lipid peroxidation. The oxalate binding proteins were thought to modulate the crystallisation process in an hyperoxaluric condition similar to calcium specific binding protein modulators.     

High calcium concentration and calcium oxalate crystals cause significant inaccuracies in the measurement of urinary osteopontin by enzyme linked immunosorbent assay

Strong evidence that osteopontin (OPN) is a determinant of urolithiasis has prompted studies comparing the protein's urinary excretion in healthy subjects and stone formers. However, reported mean urinary values have varied widely, from <1 mug/mL to more than 20 times that value. Since OPN binds to CaOx crystals, the presence of crystals in urine may cause underestimation of its urinary levels. Using a commercial ELISA, we measured urinary OPN levels in the presence of endogenous or exogenous CaOx monohydrate (COM) and dihydrate (COD) crystals. OPN concentrations decreased in the presence of endogenous and exogenous CaOx crystals, but never below 2 mug/mL. Increasing the urinary calcium concentration decreased detectable OPN levels, possibly as a result of changes in the three-dimensional conformation of the protein. Because calcium concentration and the formation of CaOx crystals cannot be controlled in urine, the use of urinary OPN levels as a biomarker for any human pathology must be seriously questioned, but particularly for the investigation of stone formers in whom hypercalciuria and crystalluria are more common than in healthy subjects.     

Measurement of the risk of calcium oxalate crystallization in urine

Summary The risk of calcium crystallization (CaOx-CR) in urine was analyzed by means of crystal counting following standardized addition of oxalate. CaOx-CR was determined in 24h urine samples from 21 stone formers and 26 normal subjects following dilution of urine to a creatinine concentration of 5 mol per ml. The mean (±SD) CaOx-CR was in stone formers 1.42±0.57 and in normal subjects 1.29±0.40. CaOx-CR was also analyzed in 16 fresh urine samples diluted to 80 per cent of the original concentration whereby values between 0.36 and 3.6 were recorded. There was a good correlation between CaOx-CR and estimates of the ion-activity product of CaOx, both in urine diluted to 5 mol of creatinine per ml and in 80 per cent diluted urine. It ist suggested that the method described is of value for evalution and follow up of patients with CaOx urolithiasis.     

Oxalate transport and calcium oxalate renal stone disease

Hyperoxaluria is considered to play a crucial role in calcium oxalate (CaOx) renal stone disease. The amount of oxalate excreted into the urine depends on intestinal absorption, endogenous production, renal clearance and renal tubular transport. Since a primary disorder has not been found so far in most CaOx stone formers and since oxalate is freely filtered at the glomerulus, most studies are presently focussed on alterations in epithelial oxalate transport pathways. Oxalate can be transported across an epithelium by the paracellular (passive) and transcellular (active) pathway. Oxalate transport across cellular membranes is mediated by anion-exchange transport proteins. A defect in the structure of these transport proteins could explain augmented transcellular oxalate transport. Little is known about the physiological regulation of oxalate transport. In this review cellular transport systems for oxalate will be summarized with special attention for the progress that has been made to study oxalate transport in a model of cultured renal tubule cells. Better understanding of the physiological processes that are involved in oxalate transport could yield information on the basis of which it might be possible to design new approaches for an effective treatment of CaOx stone disease.     

Inhibition of calcium oxalate crystallization in urine

Summary Chromatographic separation of urine showed inhibition of calcium oxalate (CaOx)-crystallization among substances with both large and small molecular weights. Ultrafiltration showed that approximately 80 per cent of the inhibiting activity, as determined in 2 per cent urine, orginated from substances with a molecular weight above 1,000. Dialysed urine was diluted to 7.5 mmol of creatinine per 1 and supersaturated with respect to CaOx. The rate of crystallization in these samples was slower in normal subjects than in stone formers (P< 0.05). The inhibiting activity in diluted urine from the two groups did not differ and neither did the concentration of alcian blue precipitable polyanions. From measurements in diluted urine it was apparent that inhibition was demonstrable with a urine concentration as low as 0.3 per cent.