Biochemical and Hematological Correlates of Elevated Homocysteine in National Surveys and a Longitudinal Study of Urban Adults.

Elevated blood homocysteine (Hcy) among middle-aged adults can increase age-related disease risk, possibly through other biochemical and hematological markers. We selected markers for hyperhomocysteinemia among middle-aged adults, studied time-dependent Hcy-marker associations and computed highly predictive indices of hyperhomocysteinemia, with cross-sectional and longitudinal validations. We used data from the National Health and Nutrition Examination Survey (NHANES III, phase 2, nmax = 4000), the NHANES 1999-2006 (nmax = 10,151) and pooled NHANES (cross-sectional validation). Longitudinal validation consisted of mixed-effects linear regression models (Hcy predicting markers' annual rates of change), applied to the Healthy Aging in Neighborhoods of Diversity Across the Life Span (HANDLS, n = 227-244 participants, k = 2.4 repeats/participant, Agebase: 30-65 years) data. Machine learning detected nine independent markers for Hcy > 14 µmol/L (NHANES III, phase 2): older age; lower folate and B-12 status; higher serum levels of creatinine, uric acid, alkaline phosphatase, and cotinine; mean cell hemoglobin and red cell distribution widths (RDW); results replicated in the 1999-2006 NHANES [AUC = 0.60-0.80]. Indices combining binary markers increased elevated Hcy odds by 6.9-7.5-fold. In HANDLS, first-visit Hcy predicted annual increase in creatinine, RDW and alkaline phosphatase, with third-visit index (2013-2018) directly predicting Hcy (2004-2009). We provide evidence of the internal and external validity of indices composed of several biomarkers that are strongly associated with elevated Hcy.


Homocysteine
Homocysteine concentrations were measured as part of an NHANES III surplus sera project on serum samples from participants 12 years of age or older who were seen during phase II of this survey (1991)(1992)(1993)(1994)). This project is described in greater detail elsewhere [1]. Homocysteine concentrations were measured at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University by using the high-performance liquid chromatography method of Araki and Sako [2]. The interassay coefficient of variation for this assay was 6%.
In NHANES III, a manual differential was performed on a special subsample of examinees aged one year and older. This manual differential was used for internal quality control purposes and to confirm abnormal hematology results. This subsample was defined as a random 10-percent sample of all examined persons plus all examinees who had a predetermined high or low value for one or more of the following hematologic assessments: white blood cell count (WBC), red blood cell count (RBC), hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet count, mean platelet volume (MPV), lymphocyte percentage, mononuclear percentage, or granulocyte percentage. A table of predetermined high and low values for WBC, RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, RDW, platelet count, MPV, lymphocyte percentage, mononuclear percentage, and granulocyte percentage is located in the Manual for Medical Technicians [3].
LUP: Serum lutein/zeaxanthin The lower limit of detection (LOD) for lutein/zeaxanthin was 0.43 ug/dL. Using the LOD coding formula (detection limit divided by the square root of two), the calculated value indicating that the serum lycopene results were below the level of detection would be 0.30. After rounding, the value of 0 (zero) was placed in the results field to indicate that the serum lutein/zeaxanthin was below 0.43 ug/dL.
LYP: Serum lycopene The lower limit of detection (LOD) for lycopene was 0.63 ug/dL. Using the LOD coding formula (detection limit divided by the square root of two), the calculated value indicating that the serum lycopene results were below the level of detection would be 0.44. After rounding, the value of 0 (zero) was placed in the results field to indicate that the serum lycopene was below 0.63 ug/dL. SFP: Serum iron This value was obtained from the standard battery of biochemical assessments. Use of the laboratory test result from the reference method (FEP), rather than the SFP value, is generally recommended. For most analyses of serum iron, the appropriate variable to use will be FEP. The value from the biochemistry profile (SFP) should not be used routinely. Consult the Laboratory Procedures Used for NHANES III (U.S. DHHS, 1996) for details. Laboratory test results for SFP were added to the protocol after NHANES III began. This result field was blank-filled for examinees who were examined prior to the start of testing.
VCP: Serum vitamin C For NHANES III, serum concentrations of vitamin C were measured using a total vitamin C, fully reduced method using high-performance liquid chromatography with electrochemical detection (HPLC-EC) analysis.

VEP: Serum vitamin E
The vitamin E value of 9999 was confirmed.

Thyroid hormones
Total (protein-bound and free) circulating thyroxine (T4) concentrations were determined using an enzyme-based homogeneous immunoassay on the Hitachi 704. The reference range is 4.5 to 12.5 μg/dL. The coefficient for inter-assay coefficient of variation (CV) was less than 10%. [4] The thyroid stimulating hormone (TSH, in mu/L) assay is a chemiluminescence immunometric assay utilizing a mouse monoclonal antibody to TSH immobilized on a polystyrene bead and a goat polyclonal antibody to TSH conjugated with an acridinium ester. The reference range is 0.3 to 5.0 mu/L. The coefficient for inter-assay CV was less than 5%. [4] Serum lipids: total cholesterol (TC), triglycerides (TG) and high-density lipoprotein-Cholesterol (HDL-C) Cholesterol is measured enzymatically in serum or plasma in a series of coupled reactions that hydrolyze cholesterol esters and oxidize the 3-OH group of cholesterol. Triglycerides are measured using a series of coupled reactions in which triglycerides are hydrolyzed to produce glycerol. HDL-cholesterol is measured following the precipitation of the other lipoproteins with a polyanion/divalent cation mixture. [4] CHP: Serum cholesterol This value was obtained from the standard battery of biochemical assessments. Use of the laboratory test result from the reference method (TCP), rather than the CHP value, is generally recommended.For most analyses of serum cholesterol, the appropriate variable to use will be TCP. The value from the biochemistry profile (CHP) should not be used routinely. Consult the Laboratory Procedures Used for NHANES III (U.S. DHHS, 1996) for the details.
LCP: Serum LDL cholesterol calculation The value for LDL was calculated by the Friedewald equation as follows: LDL = total cholesterol -high density cholesterol -triglyceride/5. Because the equation is not valid when serum triglyceride values exceed 400 mg/dL, the LDL is missing when serum triglyceride (TGP) exceeds 400 mg/dL. Serum LDL was calculated on examinees who were instructed to fast (ages 12 and older) and who did fast at least nine hours, were examined in the morning, and were randomly assigned to the morning fasting sample (WTPFHSD6 > 0). Therefore, LDL would be blank if examinees were aged less than 12 years, fasted fewer than nine hours, were examined in an afternoon or evening session, or were not randomly assigned to the morning session. For the purpose of this calculation, the number of hours fasted was rounded to the nearest whole integer. For more information regarding this equation, refer to the Laboratory Procedures Used for NHANES III (U.S. DHHS, 1996).
TGP: Serum triglycerides Serum triglyceride levels were measured regardless of the examinee's fasting status. Mean serum triglycerides and the distribution of serum triglycerides should be estimated only on examinees who did fast at least nine hours, were examined in the morning, and were randomly assigned to the morning fasting sample (WTPFHSD6 > 0). For the purpose of this calculation, the number of hours fasted was rounded to the nearest whole integer. Consult the Laboratory Procedures Used for NHANES III (U.S. DHHS, 1996) for details.

TRP: Serum triglycerides
This value was obtained from the standard battery of biochemical assessments. Use of the laboratory test result from the reference method (TGP), rather than the TRP value, is generally recommended. For most analyses, the appropriate variable to use is TGP. The value from the biochemistry profile (TRP) should not be used routinely. Consult the Laboratory Procedures Used for NHANES III (U.S. DHHS, 1996) for details. Results for TRP were added to the protocol after NHANES III began. This result field was blankfilled for examinees who were examined prior to the start of testing.

Glucose biomarkers
Serum C-peptide (first venipuncture) The specimen for this assay was obtained at the time of the initial venipuncture. This result is available for all six years of the survey. Examinees aged 40-74 years who used insulin were excluded from the OGTT. A first venipuncture was obtained, but the glucose challenge and second venipuncture were canceled. In these instances, the variables G1P, C1P and I1P have a value, but the results G2P, C2P and I2P from the second venipuncture are blank-filled to indicate a medical exclusion.
C2P: Serum C-peptide (second venipuncture) Post-glucose challenge levels of C-peptide and insulin for examinees who had an OGTT were measured only during 1991-1994.
G1P: Plasma glucose (first venipuncture) Plasma glucose was measured using the reference method on examinees aged 20 years and older. Consult the Laboratory Procedures Used for NHANES III (U.S. DHHS, 1996) for details. During NHANES III, OGTT testing was conducted on MEC examinees aged 40-74 years. A random assignment was made prior to conducting the OGGT to determine who should receive a morning examination [3,10]. As a result, approximately half of the OGGT examinees received the morning OGTT after an overnight fast. This subsample most closely conformed to the World Health Organization (WHO) criteria for OGTT testing to identify diabetes [11]. Therefore, this morning subsample is the NHANES III subsample that should be used to estimate the prevalence of diabetes and impaired glucose tolerance. People who reported a medical history of diabetes but who were not using insulin therapy were asked to conform to the fasting instructions for their examination session and were eligible for an OGTT if the age criteria were satisfied. The morning sample weights (WTPFHSD6) for total NHANES III weights for the morning OGTT subsample should be used when weighting these data to generate national estimates. Data from the afternoon and evening OGTTs do not conform to the WHO protocol for diagnosing diabetes or IGT and should not be used for these purposes.
If an examinee was given an OGTT during an examination session other than the session assigned, that examinee's sample weight for the assigned session will be zero. For example, if an examinee was selected for a morning OGTT but was tested in the afternoon, the examinee's morning sample weight for the OGTT will be zero.
GHP: Glycated hemoglobin (HbA1c) Glycohemoglobin measurements for NHANES III were performed by the Diabetes Diagnostic Laboratory at the University of Missouri --Columbia using the Diamat Analyzer System (Bio-Rad Laboratories, Hercules, CA). This ion-exchange HPLC system measures HbA1c (a specific glycohemoglobin) and has demonstrated excellent, long-term precision (interassay CV's 2.0). It was standardized to the reference method that was used for the Diabetes Control and Complications Trial (DCCT). Variant hemoglobins, including hemoglobin C, D, F, and elevated HbF, can interfere with HbA1c measurement by the Diamat HPLC. Hemoglobin S in its heterozygous state does not interfere with this assay. Although interferences usually can be detected by an abnormal Diamat chromatogram, HbA1c results for these specimens were not considered valid. Therefore, samples containing hemoglobin variants or elevated HbF or samples that produce chromatograms indicating hemoglobin degradation were analyzed by an alternate method that used affinity chromatography to separate glycohemoglobin. Affinity chromatographic methods were not affected by the presence of hemoglobin variants and were less sensitive to hemoglobin degradation due to improper sample handling. The affinity method used also was standardized to the DCCT reference method. Reasons for using the affinity method for an examinee's test included an extra peak on the chromatogram, hemoglobin C, elevated hemoglobin F, or other abnormal hemoglobin. The following steps were used to make the adjustment:

Protein biomarkers
GBP: Serum globulin Globulin results were added to the protocol after NHANES III began. This result field was blank-filled for examinees who were examined prior to the start of testing.

HGP: Hemoglobin
In NHANES I, NHANES II, and HHANES, determinations of red and white blood cell counts were made using a semiautomated cell counter Coulter model FN). Determinations of hemoglobin concentration (Hb) were made using a Coulter hemoglobinometer, and determinations of packed cell volume (PCV) were made using the microhematocrit centrifuge method. The hematologic indices MCH, MCHC, and MCV were calculated as follows:

MCH = Hb/RBC MCHC= Hb/PCV MCV = PCV/RBC
In NHANES III, these hematologic parameters were determined by using a fully automated Coulter S+JR hematology analyzer. These analyzers measured the mean (red) cell volume (MCV) directly, utilizing a process of continuous integration of pulse heights divided by the pulse number; PCV values were calculated through the multiplication of MCV and RBC.
Although it has been shown that identified errors in the microhematocrit method caused by plasma trapping and red cell dehydration approximately compensate each other, [12] packing errors can occur in macrocytic anemia and can be considerable in sickle cell anemia, spherocytosis, and thalassemias [13]. Therefore, individual values for MCV, PCV ("hematocrit"), and MCHC from NHANES III cannot be compared directly to values from the previous NHANES.

HTP: Hematocrit
See note for HGP.

MCPSI: Mean cell hemoglobin
See note for HGP.

MHP: Mean cell hemoglobin concentration
See note for HGP.

CEP: Serum Creatinine
The Cleveland Clinic Foundation (CCF) laboratory analyzed the serum creatinine specimens using a Roche coupled enzymatic assay (creatininase, creatinase, sarcosine oxidase, kits # 1775677 and 1775766) performed on a Roche P Module instrument. The Roche method calibrators were traceable to an isotope dilution mass spectrometric method for serum creatinine using standard references methods (NIST SRM 967) and confirmed by analysis of CAP LN-24 linearity set based on NIST assigned values. Serum creatinine by the Roche method was then compared to the original NHANES III measurements which used the Jaffe kinetic alkaline picrate method performed on a Roche Hitachi 737 analyzer. There were significant differences in results between these two measurements. TOP: Serum toxoplasmosis antibody The presence and quantity of antibody to Toxoplasma gondii in the test sample were determined by comparing the optical density of the test sample to a standard curve. A standard curve was constructed using optical density readings from positive control sera obtained from a kit; these readings were calibrated to WHO Toxo 60 serum and read as International Units (IU/mL). Those test samples exhibiting titer below 7 IU/mL indicated a non-significant level of antibody according to this technique; thus, they were considered to be negative, indicating no infection. Those test samples with results greater than 6 IU/mL were considered to be positive, indicating infection at some undetermined time.

(a) Alkaline phosphatase (ALP)
The method is linear up to 1000 U/L. When reanalyzing any specimen with a concentration greater than 1000 U/L, prepare a 10-fold (1+9) dilution of the specimen with saline. The minimum detection limit, based on a linear regression curve of certified material analyzed 20 times, is 2 U/L. Results below the detection limit are reported as <2 U/L.

(b) Alanine aminotransferase (ALT)
The method is linear up to 400 U/L. When reanalyzing any specimen with a concentration greater than 400 U/L, prepare a 10-fold (1+9) dilution of the specimen with saline. The minimum detection limit, based on a linear regression curve of certified material analyzed 20 times, is 1 U/L. Results below the detection limit are reported as <1 U/L.

(c) Aspartate aminotransferase (AST)
The method is linear up to 800 U/L. When reanalyzing any specimen with a concentration greater than 800 U/L, prepare a 10-fold (1+9) dilution of the specimen with saline. The results must then be multiplied by 10 to account for this dilution. The minimum detection limit, based on linear a regression curve of certified material analyzed 20 times, is 1 U/L. Results below the detection limit are reported as <1 U/L. preincubation of a combined buffer and serum solution to allow side reactions with NADH to occur, 4) substrate start (α -ketoglutarate), and 5) optimal pyridoxal phosphate activation.

(e) Gamma glutamyl transaminase (γ-GT)
The method is linear up to 1200 U/L. When reanalyzing any specimen with a concentration greater than 1200 U/L, prepare a 10-fold (1+9) dilution of the specimen with saline. The minimum detection limit, based on a linear regression curve of certified material analyzed 20 times, is 2 U/L. Results below the detection limit are reported as <2 U/L.

(f) Lactate dehydrogenase (LDH)
The method is linear up to 1000 U/L. When reanalyzing any specimen with a concentration greater than 1000 U/L, prepare a 10-fold (1+9) dilution of the specimen with saline. The minimum detection limit, based on a linear regression curve of certified material analyzed 20 times, is 2 U/L. Results below the detection limit are reported as <2 U/L.

Urinary biomarkers
Urinary iodine (IU) is measured by using the reduction-oxidation reaction between ceric and arsenite catalyzed by iodide. Therefore, UI is proportional to its catalytic activity. Urine samples, controls, and iodate standard are digested with chloric acid, then measured spectrometrically at 420 nm with a Technicon AutoAnalyzer. Calculations are based on an iodine standard curve. The final concentration of UI is expressed in μg/dL and is easily converted to μg UI per gram urinary creatinine. [4,14,15] Urinary creatinine was analyzed with a Jaffé rate reaction using an ASTRA analyzer. [4] Creatinine is released into the plasma at a relatively constant rate and thus has a constant amount per unit muscle mass. Consequently, creatinine is the best indicator of impaired kidney function.
A solid-phase fluorescent immunoassay (FIA) was used for the measurement of urinary albumin. [16] Increased microalbuminuria is a sign of renal disease and is predictive of nephropathy risk in type 2 diabetes patients. [4,16] Cadmium analysis is used to identify toxicity. Occupational exposure is the most common cause of elevated cadmium levels. [4] Cadmium is measured in urine by atomic absorption spectrometry by using a modification of the method described by Pruszkowska et al. [17] URP: Urinary creatinine Although the laboratory method detection limit for urinary creatinine is 1 mg/dL, all values below 10 mg/dL were considered "statistically suspect" and were coded as "below level of detection".

SUPPLEMENTAL METHODS 2: BIOCHEMICAL AND HEMATOLOGICAL INDICES SELECTED FOR NHANES 1999-2006
Total homocysteine (tHcy) Total homocysteine (tHcy) in plasma is measured by the "Abbott Homocysteine (HCY) assay", [18] a fully automated fluorescence polarization immunoassay (FPIA) from Abbott Diagnostics performed on the AxSYM® platform. [19] In brief, dithiothreitol (DTT) reduces homocysteine bound to albumin and to other small molecules, homocystine, and mixed disulfides, to free thiol. S-adenosyl-homocysteine (SAH) hydrolase catalyzes conversion of homocysteine to SAH in the presence of added adenosine. In the subsequent steps, the specific monoclonal antibody and the fluoresceinated SAH analog tracer constitute the FPIA detection system. [20] Plasma total homocysteine concentrations are calculated by the Abbott AxSYM® using a machine-stored calibration curve.
An international round robin performed in 1998 [21] demonstrated that the Abbott method is fully equivalent to other most frequently used methods in this field (i.e., HPLC-FD, HPLC-ED, GC/MS). Thus, the Abbott Homocysteine (HCY) assay will be used as primary method for the determination of plasma total homocysteine in NHANES 1999+. For NHANES 1999-2001, the Abbott IMx® analyzer was used, starting NHANES 2002, the Abbott AxSYM® analyzer is used. The IMx® and the AxSYM® platforms are both using the same reagent kit, but the AxSYM® is a newer fully-automated analyzer that can measure multiple analytes during one run. Pernet et al. showed that the two platforms agree well. [19] This method is linear for homocysteine in the range 0.8-50 μmol/L. Samples with results <2 μmol/L or >15 μmol/L are reanalyzed for confirmation before results are released. Samples with total homocysteine concentrations >50 μmol/L are diluted 10-fold with PBS or FPIA buffer and reanalyzed. This method has a total coefficient of variation in the range of 3-6%.

Folate/Vitamin B12 (Serum and whole blood)
Both vitamins are measured by using the Bio-Rad Laboratories "Quantaphase II Folate/vitamin B12" radioassay kit [22,23]. The assay is performed by combining serum or a whole blood hemolysate sample with 125 I-folate and 57 Co-vitamin B12 in a solution containing dithiothreitol (DTT) and cyanide. The mixture is boiled to inactivate endogenous folate-binding proteins and to convert the various forms of vitamin B12 to cyanocobalamin. The reduced folate and its analogs are stabilized by DTT during the heating. The mixture is cooled and then combined with immobilized affinity-purified porcine intrinsic factor and folatebinding proteins. The addition of these substances adjusts and buffers the pH of the reaction mixture to 9.2. The reaction mixture is then incubated for 1 hour at room temperature.

Creatinine
The LX20 modular chemistry side uses the Jaffe rate method (kinetic alkaline picrate) to determine the concentration of creatinine in serum, plasma, or urine. A precise volume of sample is introduced into a reaction cup containing an alkaline picrate solution. Absorbance readings are taken at both 520 nm and 560 nm. Creatinine from the sample combines with the reagent to produce a red color complex. The observed rate measurement at 25.6 seconds after sample introduction has been shown to be a direct measure of the concentration of the creatinine in the sample.

Uric Acid
The LX20 uses a timed endpoint method to measure the concentration of uric acid in serum, plasma, or urine. Uric acid is oxidized by uricase to produce allatoin and hydrogen peroxide. The hydrogen peroxide reacts with 4-aminoantipyrine (4-AAP) and 3.5-dichloro-2-hydroxybenzene sulfonate (DCHBS) in a reaction catalyzed by peroxidase to produce a colored product. The system monitors the change in absorbance at 520 nm at a fixed time interval. The change in absorbance is directly proportional to the concentration of uric acid in the sample.

Complete Blood Count (CBC) with Five-Part Differential NHANES 2005-2006
The Beckman Coulter method of sizing and counting particles uses measurable changes in electrical resistance produced by nonconductive particles suspended in an electrolyte.
A suspension of blood cells passes through a small orifice simultaneously with an electric current. A small opening (aperture) between electrodes is the sensing zone through which suspended particles pass. In the sensing zone, each particle displaces its volume of electrolyte. Beckman Coulter measures the displaced volume as a voltage pulse, the height of each pulse being proportional to the volume of the particle.
The quantity of suspension drawn through the aperture is for an exact reproducible volume. Beckman Coulter counts and sizes individual particles at a rate of several thousand per second. This method is independent of particle shape, color, and density.
The MAXM is a quantitative, automated, differential cell counter for in vitro diagnostic use.

Alkaline phosphatase
The DxC800 system uses a kinetic rate method using a 2-Amino-2-Methyl-1-Propanol (AMP) buffer to measure ALP activity in serum or plasma. In the reaction, the ALP catalyzes the hydrolysis of the colorless organic phosphate ester substrate, p-Nitrophenylphosphate, to the yellow colored product p-Nitrophenol and phosphate. This reaction occurs at an alkaline pH of 10.3. The system monitors the rate of change in absorbance at 410 nm over a fixed-time interval. This rate of change in absorbance is directly proportional to the ALP activity in the serum. (URL: https://wwwn.cdc.gov/Nchs/Nhanes/2011-2012/BIOPRO_G.htm)

Cotinine
Serum specimens and urine specimens are processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, and Centers for Disease Control and Prevention for analysis.
Vials are stored under appropriate frozen (-20°C) conditions until they are shipped to National Center for Environmental Health for testing.
Cotinine is a major metabolite of nicotine that may be used as a marker for both active smoking, and as an index to Environmental Tobacco Smoke (ETS) exposure, or "passive smoking". Cotinine is generally preferred over nicotine for such assessments because of its substantially longer half-life. The half-life of cotinine in plasma has been estimated to be about 15-20 hrs; by contrast, the half-life of nicotine is only 0.5-3 hrs. Cotinine may be measured in serum, urine or saliva -the half-life of cotinine in all three fluids is essentially the same. Cotinine concentrations tend to be higher (3-8x) in urine than in serum; however, for studies requiring a quantitative assessment of exposure, plasma or serum is regarded as the fluid of choice. Therefore, serum was chosen for NHANES cotinine analyses.
Serum cotinine is measured by an isotope dilution-high performance liquid chromatography / atmospheric pressure chemical ionization tandem mass spectrometry (ID HPLC-APCI MS/MS). Briefly, the serum sample is spiked with methyl-D3 cotinine as an internal standard, and after an equilibration period, the sample is applied to a basified solid-phase extraction column. Cotinine is extracted off the column with methylene chloride, the organic extract is concentrated, and the residue is injected onto a short, C18 HPLC column. The eluant from these injections is monitored by APCI-MS/MS, and the m/z 80 daughter ion from the m/z 177 quasi-molecular ion is quantitated, along with additional ions for the internal standard, external standard, and for confirmation. Cotinine concentrations are derived from the ratio of native to labeled cotinine in the sample, by comparisons to a standard curve.
NNAL is measured by using liquid chromatography linked to tandem mass spectrometry (LC/MS/MS). For "total" NNAL assays, the urine sample is fortified with an NNAL-13 C6 internal standard, and then hydrolyzed using β-glucuronidase in incubations for at least 24 hours. The samples are then extracted and cleaned up on a specially-designed solid-phase molecularly-imprinted polymer (MIP) column, after which the analyte is eluted and analyzed by LC/MS/MS, monitoring the m/z 210->180 native, and m/z 216->186 internal standard transition ions. NNAL concentrations are derived from the ratio of the integrated peaks of native to labeled ions by comparison to a standard calibration curve. Free NNAL measurements are conducted in a similar manner, but with the omission of prior enzymatic hydrolysis. Bound NNAL (i.e. NNAL-Gluc) may be estimated from the difference of (Total NNAL -Free NNAL). This method has been described previously [24].
There were no changes (from the previous 2 years of NHANES) to equipment, lab methods, or lab site. Detailed instructions on specimen collection and processing can be found in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM).

Serum homocysteine
Serum homocysteine was measured on a sub-sample of HANDLS at visit 1 using a standard enzyme immunoassay conducted at the National Institute on Aging, Clinical Research Branch Core Laboratory, that is comparable to the method used for NHANES 1999 onwards.

Serum folate and vitamin B-12
Fasting blood was collected from each participant during MRV visits. Specimens in volumes of 2 mL serum were collected in small tubes and refrigerated. Serum folate and cobalamin were measured using chemiluminescence immunoassay by Quest Diagnostics, Chantilly, VA [25].

Serum creatinine
Using participant fasting venous blood specimens, baseline serum creatinine was measured at the National Institute on Aging, Clinical Research Branch Core Laboratory, using a modified kinetic Jaffe method (CREA method, Dade Dimension X-Pand Clinical Chemistry System, Siemens Healthcare Diagnostics Inc., Newark, DE) for a small group of participants (n=88); while the majority of participants (n=1,528) had baseline serum creatinine analyzed at Quest Diagnostics, Inc. by isotope dilution mass spectrometry (IDMS) (Olympus America Inc., Melville, NY) and standardized to the reference laboratory, Cleveland Clinic. While inter-assay coefficients of variation (CV) for this sample could not be calculated due to the use of only one or the other measurement of creatinine at baseline, only intra-assay CVs (mean/SD) could be estimated and those were 0.192 and 0.187 for the CREA and the IDMS methods, respectively. Serum creatinine was measured by Quest diagnostics in remaining waves.

RDW
RDW was measured by automated Coulter DXH 800 hematology analyzer as part of peripheral complete blood count (Beckman Coulter, Brea, CA), and was expressed as coefficient of variation (%) of red blood cell volume distribution. Regular calibration was performed every 3 months on the hematology analyzer and quality control was performed according to the manufacturer's recommendations. [26] There are usually two RDW measurements used for clinical purposes, namely the RDW-coefficient of variation (CV, unit: %), which we used in this study, and the RDW-Standard Deviation (SD, unit: fL) from which RDW-CV is derived. In fact, RDW-CV=RDW-SD×100/MCV, where MCV is the mean cell volume. The normal range for RDW-CV is 11.0 -15.0%. Thus, the RDW-CV (%) depends on both the width of the distribution (normal range: 40-55 fL) curve and the MCV. [27] MCH The hematologic index MCH was calculated as follows: MCH = Hb/RBC. Using electronic cell sizing/cytometry/microscopy, Hb was assayed from a sample of 1 ml of blood drawn from participants after overnight fast and refrigerated up to 6 days (Quest diagnostics).

Serum uric acid (SUA)
SUA measurements are useful in the diagnosis and treatment of renal and metabolic disorders, including renal failure, gout, leukemia, psoriasis, starvation or other wasting conditions, and in patients receiving cytotoxic drugs. Using 1 ml of fasting blood serum, uric acid was measured using a standard spectrophotometry method. The reference range for adult men is 4.0-8.0 mg/dL, whereas for women, this range is cited as 2.5-7.0 mg/dL. (http://www.questdiagnostics.com/testcenter/TestDetail.action?ntc=905) Other reference ranges were also recently suggested and depend on the menopausal status of women. Those reference ranges are based on predictive value for gout outcomes among healthy individuals and do not necessarily predict other pathologies. Thus, based on recent research evidence, a "normal" SUA value is suggested to be <6.0 mg/dL for all healthy adult individuals.

SUPPLEMENTAL METHODS 4. DESCRIPTION OF TIME-INTERVAL MIXED-EFFECTS LINEAR REGRESSION MODELS, HANDLS 2004-2018
The main multiple mixed-effects regression models can be summarized as follows: Multi-level models vs. Composite models Eq.

1.1-1.4
Where Yij is the outcome (Each biochemical or hematological marker measured at visits 1, 2 and 3) for each individual "i" and visit "j"; is the level-1 intercept for individual i; is the level-1 slope for individual i; is the level-2 intercept of the random intercept ; is the level-2 intercept of the slope ; is a vector of fixed covariates for each individual i that are used to predict level-1 intercepts and slopes and included baseline age (Agebase) among other covariates. Xija, represents the main predictor variable (Serum Hcy at visit 1); and are level-2 disturbances; is the within-person level-1 disturbance. Of primary interest are the main effects of each exposure Xa (γ0a) and their interaction with TIME (γ1a), as described in a previous methodolgical paper. [28]   Abbreviations: Hcy=Homocysteine; LASSO= least absolute shrinkage and selection operator ; NH=Non-Hispanic; NHANES=National Health and Nutrition Examination Surveys; RBC=Red blood cells.