Variations in porphobilinogen and 5-aminolevulinic acid concentrations in plasma and urine from asymptomatic carriers of the acute intermittent porphyria gene with increased porphyrin precursor excretion.
The cause of ALA accumulation is not clear. The enzyme ALA dehydratase, which catalyzes the condensation of 2 molecules of ALA to build PBG, is not a rate-limiting step in the heme biosynthetic pathway; therefore, an inhibitory effect of PBG on the enzyme has been hypothesized (4).
Monitoring PBG and ALA variations during periods of active disease or remission is usually based on measurements of the metabolites in urine by ion-exchange chromatography, a method that has been available for more than 3 decades. There have been only a few reports on measurement of PBG and/or ALA in plasma in acute porphyria (5, 6). Recently, methods have been developed for analyzing serum ALA by use of fluorometry (7) and urinary PBG and/or ALA by mass spectrometry (8-10). We used a method based on reversed-phase HPLC with mass spectrometric detection (HPLC-MS) for measuring plasma PBG and ALA and compared plasma values with urine measurements in asymptomatic carriers of the AIP gene and in healthy individuals.
Materials and Methods
AIP carriers with a known genetic defect of the PBGD gene (11) were chosen to participate in the study (n = 10; 5 men and 5 women). The mean (SD) age was 49 (15) years for men and 37 (10) years for women. The study patients were known to be in a clinically latent phase of the disease but with increased urinary excretion of PBG and ALA for several years (Porphyria Centre Sweden).
We also recruited 5 healthy persons: 3 men and 2 women [mean age, 24 (4) years]. Written informed consent was obtained from all participants, and the study was approved by the local Ethics Committee of the Karolinska Institute (Dnr 69/01), Stockholm.
Inclusion criteria for AIP carriers were (a) urinary PBG concentrations [less than or equal to] 4 times the upper reference limit, i.e., [less than or equal to] 4.8 mmol/mol creatinine (upper reference limit, 1.2 mmol/mol creatinine); (b) at least 30 days without AIP symptoms (e.g., abdominal or muscle pain), heme therapy, or other AIP-specific treatments; (c) negative pregnancy test; and (d) no clinical history or laboratory signs of liver disease or alcohol abuse. The inclusion concentration of urinary PBG was arbitrarily determined to also ensure high plasma concentrations of PBG.
The 5 healthy individuals included in the study had undergone medical examinations and routine laboratory screening that showed no abnormalities. The measures of urinary excretion of PBG and ALA for these individuals, plus erythrocyte PBGD activity, were all within reference values.
PLASMA AND URINE SAMPLING
Each study participant was admitted to the hospital early in the morning (day 1). A short catheter was inserted percutaneously into an antecubital vein in one arm and was flushed with saline. Heparinized venous samples were obtained each hour from 0800 until 1200, and then samples were obtained at 1400 and 1600. Thus, a total of 7 blood samples were obtained during 8 h. Blood samples were immediately centrifuged at 1600g for 10 min, and plasma was stored at -80[degrees]C until analyzed.
Participants emptied their urinary bladders just before 0800 in the morning. After that, during an 8-h period in the hospital, we obtained 6 urine samples at 1-h intervals, until 1200, then at 2-h intervals until 1600. Participants were asked to empty their urinary bladders completely at each sampling time point, and the time was recorded to the nearest minute. Participants left the hospital at 1600 and continued to collect urine until 0800 on day 2, when they delivered it to the hospital; thus, a 24-h urine collection was carried out. The volumes of the urine portions were measured, and aliquots were stored at -80[degrees]C until analyzed. Participants were also instructed to bring a portion of the first morning urine on day 1 (random urine sample). All samples were kept protected from light. Participants had free access to food and beverages during the trial period.
ANALYSES OF PBG AND ALA IN URINE
We measured PBG and ALA in urine by ion-exchange chromatography (12, 13) with the Bio-Rad PBG/ALA-Test. The concentrations of urinary PBG and ALA are reported as millimoles excreted per time unit or as micromoles per mole of creatinine, i.e., normalized to the creatinine concentration of the specimen (14). Urinary creatinine analysis was performed by the Jaffe method (15). The interassay variations (as CVs) for the ionexchange chromatography method, expressed per liter, were 2.3% for PBG, 3.3% for ALA, and 2.7% for urinary creatinine. The variations for PBG and ALA, expressed per mole of creatinine, were 3.5% and 4.3%, respectively. Detection limits were 1.9 [mu]mol/L for PBG and 1.8 [mu]mol/L for ALA.
ANALYSES OF PBG AND ALA IN PLASMA
We determined plasma PBG and ALA concentrations by an HPLC-MS method. Before analysis, we deproteinized the plasma by mixing 100 [micro]L of cold acetonitrile (Merck LiChrosolv[R]) with 50 [micro]L of sample. After centrifugation of this mixture at 10 000g for 8 min, we transferred 50 [micro]L of the supernatant to a clean test tube and mixed it with 50 [micro]L of ethanol (950 mL/L) and 25 [micro]L of triethylamine (Pierce; final volume ratio, 2:2:1). The samples were dried under reduced pressure. We derivatized the free amino groups of PBG and ALA with phenylisothiocyanate (PITC; Pierce) by adding 50 [micro]L of PITC reagent (ethanol-water-triethylamine-PITC, 7:1:1:1 by volume) to each dried sample to form phenylthiocarbamyl-PBG (PTC-PBG) and phenylthiocarbamyl-ALA (PTC-ALA), respectively. The reaction was allowed to proceed for 30 min at room temperature. The samples were again dried under reduced pressure and then were dissolved in 62.5 [micro]L of 200 mL/L ethanol and centrifuged at 10 000g for 5 min. The supernatants were transferred to HPLC glass vials and kept at 4[degrees]C until analysis. We then injected 25-40 [micro]L of the supernatant on a Zorbax SB-C18 column [pore size, 80 [Angstrom]; bead size, 5 [micro]m; 15 cm x 2.1 mm (i.d.); Rockland Technologies Inc.] with a Zorbax SB-C8 guard column (pore size, 300 [Angstrom]; Rockland Technologies). Both columns had been equilibrated with a 95%-5% mixture of 25 mmol/L formic acid in water (Merck; buffer A) and 25 mmol/L formic acid in acetonitrile (Merck LiChrosolv[R]; buffer B). The PTC-PBG and PTC-ALA derivatives were eluted with a 2-component gradient as follows: 2 min of 95% buffer A-5% buffer B, then via linear gradient to 40% buffer A-60% buffer B by 17 min after sample injection. This concentration ratio was maintained for 1 min before buffer A was decreased to 20% during 2 min and finally returned to the starting point (5%) after an additional 2 min. The flow rate was 0.2 mL/min. The ALA derivative eluted at 15-16 min and the PBG derivative at 18-19 min.
Sample detection was performed by selected-ion monitoring at positive polarity with an electrospray quadropole mass spectrometer (Agilent/Hewlett Packard 1100 HPLC-MSD) equipped with a binary pump system, auto-injector, and diode array detector controlled by HP-Chemstation software. The drying gas temperature was 320[degrees]C, at a flow rate of 10.0 L/min; nebulizer pressure was 25 psig; and capillary voltage was set to 4000 V. PTC-ALA ions were monitored at relative molecular masses ([M.sub.r]) of 189.1 and 249.2, and PTC-PBG ions at Mr 210.2 and 362.2 (fragmentor voltages were set to 130 and 40 V, respectively). Calibration curves were constructed each time by analysis of PBG (Sigma) and ALA (Bio-Rad) calibrators prepared by adding increasing amounts of both PBG and ALA (1.0, 2.0, 5.0, 10, 15, 20, and 30 pmol) to a human plasma sample with no detectable endogenous PBG and only trace amounts of ALA. The calibration curves for both PBG and ALA were linear in this range (R2 >0.97). The unknown samples, analyzed in duplicate, were compared with the calibration curves, and the PBG and ALA concentrations were expressed as micromoles per liter. The lower limit of detection, which represented a signal that was at least 3 SD above the background, was 1 pmol (corresponding to 0.12- 0.2 [mu]mol/L of plasma, depending on the injection volume) for both ALA and PBG. The intraassay variation (CV), calculated from the duplicate samples from the AIP carriers (n = 77), was 6.8% for PBG and 9.9% for ALA. The mean interassay CVs for PBG and ALA, established previously by measuring 3 samples on 4 different occasions within a period of 2 months, were 14% and 15%, respectively. We observed no loss of PBG or ALA in plasma kept at -80[degrees]C for 2.5 years. The absolute recovery through the analysis could not be evaluated because derivatized compounds were not available for direct injection into the mass spectrometer. Instead, we compared the work-up procedures from water and plasma. This relative recovery in plasma was 103% for ALA and 85% for PBG.
PBG AND ALA CLEARANCE
We determined the renal clearances for endogenous PBG and ALA in the 10 AIP carriers and for ALA in the 5 healthy individuals with the formula:
[C.sub.PBG (or ALA)] = [U.sub.PBG (or ALA)] x V/[P.sub.PBG (or ALA)] (mL/min)
where U and P are the urinary and plasma concentrations, and V is the urine flow rate. The clearances were calculated from the pooled 8-h urine values ([mu]mol/L) and the mean plasma concentration values ([mu]mol/L) of the 7 blood samples from each participant.
Values are reported as the mean (SD). In all calculations, the mean values include the 10 participating AIP carriers and the 5 healthy participants separately. We performed statistical analyses and graphics with Microsoft Excel[R] software with the Analyze-It[TM] (Analyze-It Software Ltd.) statistical software add-in. For pairwise comparisons, we used the Student t-test, and for association tests, we computed the Pearson correlation coefficient with a confidence interval. P values were calculated by use of the t-approximation, and a P value <0.05 was considered statistically significant. All tests were two-tailed.
The mean (SD) plasma PBG and ALA concentrations for the 10 AIP carriers were 3.1 (1.0) [mu]mol/L (range, 1.7-5.1 [mu]mol/L) and 1.7 (0.7) [mu]mol/L (range, 0.9 -3.6 [mu]mol/L), respectively. The corresponding values for the excreted amounts of urinary PBG and ALA were 102 (25) [mu]mol/8 h (range, 68-146 [mu]mol/8 h) and 56 (18) [mu]mol/8 h (range, 32-91 [mu]mol/8 h). In the 5 healthy individuals, plasma PBG concentrations were below the detection limit for the method (<0.12 [mu]mol/L), and the mean value for plasma ALA was 0.38 (0.03) [mu]mol/L (range, 0.36-0.41 [mu]mol/L). The mean values for urinary PBG and ALA in the healthy individuals were 2.9 (0.7) [mu]mol/8 h (range, 2.3- 4.1 [mu]mol/8 h) and 9.3 (1.2) [mu]mol/8 h (range, 7.8 -10.5 [mu]mol/8 h), respectively. The individual concentrations and variations for plasma and urine PBG and ALA for the AIP carriers and healthy individuals are presented in Table 1. The individual patterns in one AIP carrier with low and in another with high intraindividual variation are illustrated in Fig. 1.
The renal clearance values for PBG and ALA during 8 h are also shown in Table 1. Because of the low plasma concentrations of PBG in the healthy individuals, we could not calculate their PBG clearance. There was no statistically significant difference between the values for renal clearance of PBG and ALA in the AIP carriers (P = 0.730, paired t-test). The mean (SD) creatinine clearance for the AIP carriers was 90.4 (12.0) mL/min (range, 74-113 mL/min) for 8 h. The corresponding value for the healthy participants was 149 (25.8) mL/min (range, 119-176 mL/min). The reference interval is 90-150 mL/min for individuals up to 50 years of age. We found no correlation between creatinine clearance and PBG or ALA clearance.
[FIGURE 1 OMITTED]
The total urine excretion of PBG in 24 h was 244 (51.4) [mu]mol in AIP carriers and 6.5 (1.1) [mu]mol in healthy individuals. For ALA, the corresponding values were 136 (39.4) and 25.8 (3.7) [mu]mol, respectively. In both AIP carriers and healthy individuals, the amount of PBG and ALA excreted during the 8-h study period (Table 1) was thus ~40% of the total amount in the 24-h collection.
AIP carriers showed no statistically significant difference between urinary PBG concentrations in the random morning sample compared with samples collected during 8 or 24 h (normalized to creatinine; P = 0.257 and 0.426, respectively). The same was true for urinary ALA concentration (P = 0.203 and 0.229, respectively). The results remained unchanged when the samples obtained from healthy individuals were included.
The concentrations of both plasma and urinary PBG in the AIP carriers were twice as high as those for plasma and urinary ALA. The mean (SD) PBG/ALA ratio was 2.0 (0.8) in plasma (range, 1.2-3.3) and 2.0 (0.5) in urine (range, 1.3-3.1). In healthy individuals, the corresponding mean (SD) ratio for urinary PBG/ALA was 0.32 (0.07), and the range was 0.23- 0.41. This ratio is in the same range as the range we found for AIP carriers in the clinically and biochemically latent phase (data not shown).
The correlation between mean plasma PBG concentration and the total amount of PBG excreted in the urine over 8 h was significant at the 5% level (r = 0.678; 95% confidence interval, 0.09-0.92; P = 0.031), and between plasma and urinary ALA, it was significant at the 1% level (r = 0.856; 95% confidence interval, 0.49-0.97; P = 0.0016; Fig. 2). The healthy individuals were not included because their plasma values were below the limit of detection for the method (PBG) or were very low (ALA).
To study the general variation patterns for plasma PBG and ALA ([mu]mol/L) during the 8-h observation period, we calculated each single determination as a percentage of the mean value for the 7 plasma determinations for each AIP carrier. We performed the same procedure for the 6 urine determinations (expressed as [mu]mol/min) in each AIP carrier. The plasma PBG concentration was significantly higher in the morning sample (drawn at 0800) than in the last sample drawn at 1600 (P <0.001, paired t-test; Fig. 3). This was also true when urinary PBG concentrations measured in samples collected between 0800 and 0900 were compared with those collected between 1400 and 1600 (P <0.002). We observed no differences in plasma or urinary ALA concentrations when we compared the morning and afternoon samples.
In the asymptomatic AIP carriers, the plasma concentrations of PBG and ALA quantified by the described HPLC-MS method showed high correlation over time with the urinary concentrations for both mean values (Fig. 2) and individual values (Table 1 and Fig. 1). These facts together with the fact that the PBG/ALA ratio in plasma was ~2 for AIP carriers, the same as found for urine, confirm the accuracy of the described method for plasma.
[FIGURE 2 OMITTED]
There have been only a few previous reports on plasma PBG concentrations. To measure serum PBG and ALA in AIP carriers during different stages of the disease, Miyagi et al. (6) used a modified method based on one described by Mauzerall and Granick (13). In their study (6), the patients in clinical remission had high urinary and plasma concentrations of porphyrin precursors, in accordance with our results, and the plasma concentrations were in the same range as in our AIP carriers. The mean concentrations of serum PBG (5.3 [mu]mol/L) were higher than the mean concentrations of ALA (3.3 [mu]mol/L), which is the same pattern as found in our study.
The concentrations of PBG and ALA in plasma and urine differed between the AIP carriers (Table 1) but were relatively constant within each individual during the 8 h of observation, with the exception of participant 7 (Fig. 1). In this individual, the peak-like increase in the ALA concentration was followed 1 h later by increased urinary ALA. No change in plasma PBG was observed, but there was a slight and sustained increase in urinary PBG. These observations corroborate the higher correlation found between the concentrations of ALA in plasma and urine compared with the correlations between PBG in plasma and urine. Because the renal clearances for PBG and ALA were not significantly different, the different behaviors of PBG and ALA cannot be explained by different renal clearances for these metabolites.
[FIGURE 3 OMITTED]
The clearance values for PBG and ALA were ~77% of that for creatinine in the AIP carriers, and in the healthy participants the clearance of ALA was ~34% of that for creatinine. The AIP carriers had lower creatinine clearance than the healthy participants. The finding that the renal clearance values for ALA in the AIP carriers were approximately in the same range as in the healthy individuals (Table 1) suggests that the AIP carriers had conserved enough renal function to handle the increased plasma concentration of ALA (and PBG).
In healthy participants, the PBG/ALA ratio in urine was ~0.3, which is the same ratio as found in latent AIP carriers (data not shown). In contrast, in the AIP carriers with high excretion of porphyrin precursors, the PBG/ ALA ratio was ~2.0 in both plasma and urine. This finding cannot be explained by different renal handling of the metabolites (see above); it probably indicates that the deficient PBGD enzyme may be overloaded in asymptomatic patients with high PBG and ALA excretion and may cause selective accumulation of PBG, the substrate for the enzyme. The increased ALA concentrations in AIP may reflect up-regulation of the biosynthetic pathway to compensate for heme deficiency and/or a possible inhibitory effect of PBG on ALA dehydratase (1, 4). In other studies in which the patterns for PBG and ALA have been described during symptomatic periods, it has been shown that the PBG/ALA ratio returns to reference values during remission after treatment (16, 17).
When studying the general variation patterns of plasma and urinary PBG and ALA, we found a significant difference between the morning and afternoon PBG values in plasma and urine, a decrease in PBG that may reflect circadian variation (Fig. 3). Further studies in larger groups are needed to corroborate this observation. In our study, the plasma and urinary ALA concentrations within individuals were stable during the observation period, a finding that is in accordance with that of Gorchein and Webber (5), who did not find any circadian variation of ALA during 24 h of observation in healthy individuals.
Measurement of porphyrin precursors in urine has been performed for decades. The method has reliable sensitivity and is readily performed in the clinical laboratory, but urine measurements are laborious and timeconsuming and affected by several factors, such as the pH of the sample, temperature, exposure to light, diuresis, and sampling errors. When PBG and ALA concentrations were normalized to creatinine, we found no significant differences between the values in the initial random urine sample compared with the values in the 8- or 24-h collections; we thus conclude that random samples of morning urine are preferable to time samplings.
To our knowledge, this is the first report surveying the porphyrin precursor patterns over time in plasma and urine in asymptomatic but biochemically active AIP carriers. When the HPLC-MS or -tandem MS technology becomes available in clinical laboratories, measurement of PBG and ALA in plasma may facilitate the monitoring of acute porphyria crises. Such monitoring could be particularly beneficial in the management of AIP patients with renal failure, particularly if they are in an anuric state. The analysis of PBG and ALA in plasma represents a new tool that may further understanding of the pathophysiology of acute porphyria attacks and improve treatment monitoring.
Received July 27, 2005; accepted January 24, 2006.
Previously published online at DOI: 10.1373/clinchem.2005.058198
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(4) Nonstandard abbreviations: AIP, acute intermittent porphyria; PBGD, porphobilinogen deaminase; PBG, porphobilinogen; ALA, 5-aminolevulinic acid; MS, mass spectrometry; PITC, phenylisothiocyanate; PTC-PBG, phenylthiocarbamyl porphobilinogen; PTC-ALA, phenylthiocarbamyl 5-aminolevulinic acid; and [M.sub.r], relative molecular mass.
YLVA FLODERUS,  ELIANE SARDH,  CHRISTER MOLLER,  CLAES ANDERSSON,  LILLAN REJKJAER,  DAN E.H. ANDERSSON,  and PAULINE HARPER  *
 Porphyria Centre Sweden, Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden.
 Department of Internal Medicine, Karolinska Institute, Stockholm Soder Hospital, Sweden.
 Zymenex A/S, Hillerod, Denmark, and Lidingo , Sweden.
* Address correspondence to this author at: Porphyria Centre Sweden, CMMS C2 71, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden. Fax 46-8-58582760; e-mail email@example.com.
Table 1. Individual PBG and ALA concentrations and ratios in urine and plasma, total excretion in urine, and renal clearance in 10 asymptomatic but biochemically active AIP carriers and in 5 healthy individuals. Concentration in Mean (SD) random morning concentration in 7 urine sample, plasmas, [micro] mmol/mol creatinine mol/L Study participant PBG ALA PBG ALA AIP carriers 1 18.5 11.5 2.9 (0.2) 1.5 (0.2) 2 24.8 7.4 5.1 (0.6) 1.7 (0.3) 3 16.7 10.0 2.8 (0.4) 1.6 (0.3) 4 21.2 14.9 2.1 (0.4) 1.6 (0.3) 5 23.8 9.0 3.6 (0.3) 1.4 (0.2) 6 16.6 9.8 3.0 (0.5) 0.9 (0.1) 7 27.0 11.6 3.9 (0.2) 3.6 (1.7) 8 28.7 14.6 3.4 (0.5) 2.0 (0.3) 9 20.1 11.5 2.5 (0.1) 2.0 (0.1) 10 19.4 7.9 1.7 (0.2) 1.2 (0.1) Mean (SD) 3.1 (1.0) 1.7 (0.7) Healthy individuals 1 0.3 1.8 <0.12 (a) 0.36 (0.05) 2 0.5 1.4 <0.12 0.38 (0.06) 3 0.3 1.1 <0.12 0.41 (0.05) 4 0.3 1.6 <0.12 0.38 (0.07) 5 0.4 1.4 <0.12 0.37 (0.09) Mean (SD) 0.38 (0.03) Mean (SD) concentration in 6 urine samples, mmol/mol Plasma creatinine PBG/ALA Study participant ratio PBG ALA AIP carriers 1 1.9 (0.3) 19.3 (2.0) 10.5 (0.8) 2 3.2 (0.7) 26.0 (2.6) 8.4 (0.8) 3 1.9 (0.4) 20.5 (1.8) 11.3 (2.1) 4 1.3 (0.2) 21.3 (2.6) 12.5 (1.1) 5 2.7 (0.4) 20.3 (0.8) 8.4 (1.2) 6 3.3 (0.5) 23.0 (4.3) 9.4 (1.2) 7 1.3 (0.4) 36.3 (4.2) 23.6 (9.6) 8 1.8 (0.1) 28.5 (5.0) 16.0 (1.9) 9 1.2 (0.1) 19.9 (2.0) 15.2 (2.0) 10 1.5 (0.3) 16.8 (1.0) 10.8 (0.9) Mean (SD) 2.0 (0.8) 23.2 (5.7) 12.6 (4.7) Healthy individuals 1 0.37 (0.08) 1.64 (0.17) 2 0.60 (0.13) 1.46 (0.21) 3 0.45 (0.10) 1.31 (0.11) 4 1.28 (0.50) 2.07 (0.13) 5 0.58 (0.22) 2.07 (0.29) Mean (SD) 0.54 (0.13) 1.71 (0.35) Total urinary excretion in Urine 8 h, [micro] mol PBG/ALA Study participant ratio PBG ALA AIP carriers 1 1.9 (0.3) 132 72.7 2 3.1 (0.3) 120 39.5 3 1.9 (0.4) 88.4 52.4 4 1.7 (0.2) 89 53.0 5 2.5 (0.3) 105 45.0 6 2.4 (0.3) 76.9 32.2 7 1.7 (0.6) 146 91.2 8 1.8 (0.2) 113 64.9 9 1.3 (0.1) 83.1 63.1 10 1.6 (0.1) 68.4 44.0 Mean (SD) 2.0 (0.5) 102 (25) 55.8 (17.6) Healthy individuals 1 0.23 (0.06) 2.33 10.5 2 0.41 (0.07) 4.1 10.2 3 0.34 (0.06) 2.76 8.26 4 0.34 (0.08) 2.66 7.81 5 0.28 (0.11) 2.75 9.70 Mean (SD) 0.32 (0.07) 2.92 (0.68) 9.29 (1.19) Renal clearance (8 h), mL/min Study participant PBG ALA AIP carriers 1 94.7 99.0 2 49.1 50.0 3 65.7 70.5 4 86.9 68.1 5 61.7 69.3 6 53.3 74.6 7 77.5 52.8 8 68.4 69.0 9 69.9 65.2 10 82.7 76.6 Mean (SD) 71.0 (14.6) 69.5 (13.4) Healthy individuals 1 62.5 2 54.8 3 41.6 4 41.4 5 55.1 Mean (SD) 51.1 (9.3) (a) The detection limit of the assay is 0.12 [micro] mol/L.
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|Title Annotation:||Endocrinology and Metabolism|
|Author:||Floderus, Ylva; Sardh, Eliane; Moller, Christer; Andersson, Claes; Rejkjaer, Lillan; Andersson, Dan|
|Date:||Apr 1, 2006|
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